Lung cancer differential marker

ABSTRACT

An object of the present invention is to develop and provide a lung cancer differential marker with which lung cancer can be diagnosed conveniently and highly sensitively without depending only on increase or decrease in protein expression level between cancer patients and healthy persons. Another object of the present invention is to develop and provide a glycan marker capable of distinguishing histological types of lung cancer. Of serum glycoproteins, glycopeptide and glycoprotein groups whose glycan structures were altered specifically in lung cancer cell culture supernatants were identified, and they are provided as lung cancer differential markers.

TECHNICAL FIELD

The present invention relates to a lung cancer differential markerglycoprotein having a glycan or a fragment thereof, and a method fordetermining the presence or absence of lung cancer or the histologicaltype of lung cancer using the same.

BACKGROUND ART

Lung cancer is a typical example of intractable cancers and is the firstand second leading causes of cancer deaths in Japanese men and women,respectively, in 2009. The types of lung cancer are broadly classifiedinto small cell cancer (small cell lung cancer) accounting forapproximately 10% and non-small cell cancer accounting for approximately90%. The types of non-small cell cancer are further classified intoadenocarcinoma (lung adenocarcinoma) (60%), squamous cell carcinoma(lung squamous cell carcinoma) (25%), and large-cell cancer (large-celllung cancer) (5%).

The small cell lung cancer is a very high-grade cancer and therefore hasa strong tendency to metastasize even in an early stage. As seen inprevious cases, it is likely that the small cell lung cancer has alreadymetastasized systemically when found. Thus, non-surgical therapy iscommonly selected for this cancer even if its metastasis to lymph nodesor other tissues is not confirmed. Since this cancer is highly sensitiveto chemotherapy or radiation, chemotherapy is central to thenon-surgical therapy.

By contrast, the non-small cell lung cancer, which constitutes a largeportion of lung cancer cases, is low sensitive to chemotherapy orradiation. For its treatment, it is important to find the cancerrelatively early and remove the lesion by surgical therapy.

Tests for lung cancer can be broadly classified, depending on thepurposes of the tests, into three: (1) lung cancer assessment to testthe probability of lung cancer; (2) definite diagnosis of lung cancer toconfirm that this probable lung cancer is definitely lung cancer; and(3) determination of the stage of lung cancer progression to test thehistological type and stage of progression of the definitely diagnosedlung cancer.

These tests generally adopt a method which involves detecting anabnormal shadow in the lung field by chest X-ray examination or CT scanand subsequently finally determining a cancer type and comprehensivelydetermining the stage of progression by bronchoscopy or by thepathological diagnosis of biopsy samples obtained using biopsy or thelike. However, cases with small cell cancer coexisting with non-smallcell cancer or borderline cancers might be given different diagnosticoutcomes among pathologists. Accurate definite diagnosis has not yetbeen established for lung cancer.

In recent years, tumor markers have been used in cancer prognosis or thelike. The tumor markers refer to substances produced by cancer cells orsubstances produced by cells in response to cancer cells. The amounts ofthe tumor markers contained in serum reflect the amount or histologicaltype of tumor. The tumor markers can therefore serve as an index for,for example, determining the presence or absence of cancer and as such,can be used in diagnostic aids, the prediction of a histological type orthe stage of progression, the determination of therapeutic effects, theprediction of recurrence, prognosis, etc. Currently, some tumor markers,such as CEA, CYFRA, NSE, ProGRP, SCC, and SLX, are also known for lungcancer (Non Patent Literatures 1 to 3). All of these tumor markers,however, are based on the difference in protein expression level, i.e.,increase or decrease in protein expression, in blood or tissue betweenhealthy persons and lung cancer patients. These tumor markers areusually expressed even in normal cells and are thus low specific forlung cancer. Hence, the obtained results present the problem of lowreliability or detection sensitivity. In addition, lung cancer markersuseful in determining the histological type of detected lung cancer havenot yet been found.

CITATION LIST Non Patent Literature

-   Non Patent Literature 1: Molina R, et al., (2005) Anticancer Res 25:    1773-1778.-   Non Patent Literature 2: Mizuguchi S, et al., (2007) Ann Thorac Surg    83: 216-221.-   Non Patent Literature 3: Holdenrieder S, et al., (2008) Clin Cancer    Res 14: 7813-7821.

SUMMARY OF INVENTION Technical Problem

An object of the present invention is to develop a lung cancerdifferential marker with which lung cancer can be diagnosed convenientlyand highly sensitively without depending only on increase or decrease inprotein expression level between cancer patients and healthy persons.More specifically, an object of the present invention is to develop alung cancer-specific differential marker glycoprotein and a fragmentthereof, which serve as an indication for suffering lung cancer.

Another object of the present invention is to develop a lung cancerdifferential marker capable of determining the histological type of lungcancer.

A further object of the present invention is to develop a glycan probefor differential diagnosis of lung cancer, with which the presence orabsence of lung cancer and further, its histological type can bedetermined by histological staining.

Solution to Problem

The compositional and structural diversities of glycans on proteinssecreted from cells are controlled by the balanced expression ofhundreds of glycan-related genes and vary depending on the degrees ofcell differentiation and cancer progression. Glycoproteins whose glycanstructures are altered can be used as disease condition index markersincluding tumor markers. In recent years, such glycan-related tumormarkers based on proteomics have been searched for actively. In thepipeline of the marker search, first, candidate molecules are identifiedby large-scale analysis at phase 1. Subsequently, the candidatemolecules are tested by quantitative analysis at phase 2 to narrow downthe candidates. Then, a validation test is conducted at phase 3.

In order to attain the objects, the present inventors have searched forlung cancer differential markers using glycoproteomics based on themarker search pipeline. As a result, the present inventors havesuccessfully identified, from among serum glycoproteins, novelglycoprotein or glycopeptide groups having lung cancer-specificstructures detected in a lung cancer cell culture supernatant. Thepresent inventors have also revealed that the presence or absence oflung cancer and the histological type of lung cancer can be determinedusing these glycoprotein or glycopeptide groups. The present inventionis based on these findings and provides the followings:

(1) A lung cancer differential marker glycoprotein listed in Table 1 or2, being glycosylated with a glycan at the asparagine residue(s) at theglycosylation site(s) shown in Table 1 or 2:

TABLE 1  Protein Small cell cancer Adenocarcinoma Glycosylation SEQ #Protein name gi(ID) AAL ConA AAL ConA site Peptide sequence NO. 1acid alpha-glucosidase gi|119393891, ◯ X X X 390 QVVENMTR 1 gi|119393893470 GVFITNETGQPLIGK 2 882 GAYTQVIFLARNNTIVNELVR 3 2 biotinidasegi|4557373 X X ◯ X 119 DVQIIVFPEDGIHGFNFTR 4 150 FNDTEVLQR 5 349SHLIIAQVAKNPVGLIGAENATGETDPSHSK 6 349 NPVGLIGAENATGETDPSHSKFLK 7 3cathepsin D gi|4503143 X X ◯ X 263 YYKGSLSYLNVTR 8 4 cathepsin L1gi|22202619, ◯ X X X 221 YNPKYSVANDTGFVDIPKQEK 9 gi|4503155 221YSVANDTGFVDIPK 10 5 cathepsin L2 gi|23110960 ◯ X X X 221YRPENSVANDTGFTVVAPGKEK 11 292 NLDHGVLVVGYGFEGANSNNSK 12 6cell adhesion molecule 4 gi|21686977 ◯ X X X 67 QTLFFNGTR 13 7deoxyribonuclease II, gi|4503349 ◯ X X X 86 SNTSQLAFLLYNDQPPQPSK 14lysosomal 8 fibronectin 1 gi|47132557 (isoform 1), X X ◯ X 430GGNSNGALCHFPFLYNNHNYTDCTSEGR 15 gi|47132551 (isoform 2), 528DQCIVDDITYNVNDTFHK 16 gi|16933542 (isoform 3), 542 RHEEGHMLNCTCFGQGR 17gi|47132555 (isoform 4), 542 HEEGHMLNCTCFGQGR 18gi|47132553 (isoform 5), 1007 ESKPLTAQQTTKLDAPTNLQFVNETDSTVLVR 19gi|47132549 (isoform 6), 1007 LDAPTNLQFVNETDSTVLVR 20gi|47132547 (isoform 7) 1291 WTPLNSSTIIGYR 21 9 galectin-3-bindinggi|122937327 ◯ X X X 44 ADVGGEAAGTSINHSQAVLQR 22 protein-like 61QGNASDVVLR 23 307 FFDVNGSAFLPR 24 10 insulin-like growth gi|119964726 ◯X X X 112 SLLEFNTTVSCDQQGTNHR 25 factor 2 receptor 435 MSVINFECNKTAGNDGK26 582 TNITLVCKPGDLESAPVLR 27 2085 GYPCGGNKTASSVIELTCTK 28 11insulin-like growth factor gi|62243248 (isoform a), X X ◯ X 116GLCVNASAVSR 29 binding protein 3 gi|62243068 (isoform b) 205YKVDYESQSTDTQNFSSESKR 30 12 insulin-like growth factor gi|56090548 X ◯ XX 166 DGPCEFAPVVVVPPRSVHNVTGAQVGLSCEVR 31 binding protein-like 1 166SVHNVTGAQVGLSCEVR 32 13 integral membrane protein 1 gi|22749415 X ◯ X X548 TILVDNNTWNNTHISR 33 14 L1 cell adhesion moleculegi|4557707 (isoform 1), X X ◯ ◯ 433 ILTADNQTYMAVQGSTAYLLCK 34gi|13435353 (isoform 2) 671 WYSLGKVPGNQTSTTLK 35 777VQWRPQGTRGPWQEQIVSDPFLVVSNTSTFVPYEIK 36 979 THNLTDLSPHLR 37 15lysosomal acid phosphatase 2 gi|4557010 ◯ X X X 133FNPNISWQPIPVHTVPITEDR 38 167 YEQLQNETRQTPEYQNESSR 39 177 QTPEYQNESSR 4016 melanoma cell adhesion gi|71274107 ◯ X X X 56 CGLSQSQGNLSHVDWFSVHK 41molecule 418 CVASVPSIPGLNR 42 17 melanoma-associated antigengi|134244281 (isoform 1), ◯ ◯ X X 38 WCATSDPEQHKCGNMSEAFR 43 p97gi|16163666 (isoform 2) 515 DCDVLTAVSEFFNASCVPVNNPK 44 18neogenin homolog 1 gi|4505375 X ◯ X X 73 GSSVILNCSAYSEPSPK 45 210VIKLPSGMLVISNATEGDGGLYR 46 470 TPASDPHGDNLTYSVFYTK 47 19neural cell adhesion gi|94420689 (isoform 1), ◯ X X X 347 TSTRNISSEEK 48molecule 1 gi|115529482 (isoform 2), 449 DGQLLPSSNYSNIK 49gi|115529478 (isoform 3) 478 IYNTPSASYLEVTPDSENDFGNYNCTAVNR 50 20neuronal pentraxin 11 gi|28195384 ◯ X X X 148 ANVSNAGLPGDFR 51 189VAELEDEKSLLHNETSAHR 52 21 neuronal pentraxin receptor gi|17402888 X ◯ XX 42 ALPGGADNASVASGAAASPGPQR 53 22 ribonuclease T2 gi|5231228 X X ◯ X106 AYWPDVIHSFPNR 54 212 QDQQLQNCTEPGEQPSPK 55 23 secretogranin IIIgi|19557645 ◯ ◯ X X 68 KTYPPENKPGQSNYSFVDNLNLLK 56 346 NKLEKNATDNISK 5724 sel-1 suppressor of gi|19923669 X ◯ X X 608 EASIVGENETYPR 58lin-12-like 25 sparc/osteonectin, cwcv and gi|7662036 ◯ ◯ X X 225LRDWFQLLHENSKQNGSASSVAGPASGLDK 59 kazal-like domains 225QNGSASSVAGPASGLDK 60 proteoglycan (testican) 2 26Thy-1 cell surface antigen gi|19923362 ◯ ◯ X X 42 LDCRHENTSSSPIQYEFSLTR61 27 tubulointerstitial gi|11545918 X X ◯ X 78 GRADDCALPYLGAICYCDLFCNR62 nephritis antigen-like 1 161 AINQGNYGWQAGNHSAFWGMTLDEGIR 63 28 v-kit Hardy-Zuckerman 4 gi|4557695 (isoform 1), ◯ ◯ X X 130SLYGKEDNDTLVR 64 feline sarcoma viral gi|148005039 (isoform 2) 367TFTDKWEDYPKSENESNIR 65 oncogene homolog 463 CSASVLPVDVQTLNSSGPPFGK 66486 LVVQSSIDSSAFKHNGTVECK 67 29 laminin alpha 5 gi|21264602 X X ◯ ◯ 95LVGGPVAGGDPNQTIR 68 921 LNLTSPDLFWLVFR 69 1330 VWQGHANASFCPHGYGCR 701529 TIPPDCLLCQPQTFGCHPLVGCEECNCSGPGIQELT DPTCDTDSGQCK 71 2019CEICAPGFYGNALLPGNCTR 72 2196 GINASSMAWAR 73 2209 LHRLNASIADLQSQLR 742303 TLSELMSQTGHLGLANASAPSGEQLLR 75 2423 DNATLQATLHAAR 76 2501LVEAAEAHAQQLGQLALNLSSIILDVNQDRLTQR 77 2568 QGLVDRAQQLLANSTALEEAMLQEQQR78 2707 GVHNASLALSASIGR 79 3107 LNTTGVSAGCTADLLVGR 80 3287VFDLQQNLGSVNVSTGCAPALQAQTPGLGPR 81 30 laminin, beta 1 gi|4504951 X X ◯ ◯1041 KCVCNYLGTVQEHCNGSDCQCDK 82 1279 LSDTTSQSNSTAK 83 1487QSAEDILLKTNATK 84 1643 AIKQADEDIQGTQNLLTSIESETAASEETLFNASQR 85 31phospholipid transfer gi|5453914 (isoform a), X X ◯ ◯ 64 GKEGHFYYNISEVK86 protein gi|33356541 (isoform b) 143 MKVSNVSCQASVSR 87 143VSNVSCQASVSR 88 245 GAFFPLTERNWSLPNR 89 398 FRIYSNHSALESLALIPLQAPLK 90193 GAFFPLTERNWSLPNR 91

TABLE 2  Protein Small cell cancer Adenocarcinoma Glycosylation SEQ #Protein name gi(ID) AAL ConA AAL ConA site Peptide sequence NO 1activated leukocyte cell gi|68163411 X ◯ X X 167 KLGDCISEDSYPDGNITWYR 92adhesion molecule 265 NAIKEGDNITLK 93 265 EGDNITLK 94 361 NATVVWMKDNIR95 480 YYSKIIISPEENVTLTCTAENQLER 96 2 alpha 2 type V collagengi|89363017 X X X ◯ 1400 EASQNITYICK 97 preproprotein 3amiloride binding gi|73486661 X X ◯ ◯ 538 LENITNPWSPR 98 protein 1 4aspartate beta-hydroxylase gi|14589866 (isoform a) X X X ◯ 452LVQLFPNDTSLKNDLGVGYLLIGDNDNAKK 99 5 aspartate beta-hydroxylasegi|14589864 (isoform b) ◯ ◯ X X 64 DFRYNLSEVLQGK 100 6beta-1,3-galactosyl-O- gi|148277029, X X ◯ X 58 HLELAGENPSSDINCTK 101glycosyl-glycoprotein gi|148277031, 95 WTPDDYINMTSDCSSFIK 102beta-1,6-N-acetyl- gi|148277033, glucosaminyltransferase gi|148277035,gi|21614523 7 bone morphogenetic gi|4502421 X X ◯ ◯ 142AATSRPERVWPDGVIPFVIGGNFTGSQR 103 protein 1 363 ISVTPGEKIILNFTSLDLYR 104599 LNGSITSPGWPK 105 8 calsyntenin 2 gi|11545861 ◯ X X X 98IHGQELPFEAVVLNKTSGEGR 106 374 NLTDQFTITMWMK 107 716 QECLELNHSELHQR 108729 HLDATNSTAGYSIYGVGSMSR 109 9 carboxypeptidase D gi|22202611 X ◯ X X172 LLNTTDVYLLPSLNPDGFERAR 110 522 RFANEYPNITR 111 978HIWSLEISNKPNVSEPEEPKIR 112 1070 GKDLDTDFTNNASQPETK 113 10 CD47 antigengi|4502673 (isoform 1), X ◯ X X 73 GRDIYTFDGALNK 114gi|38683836 (isoform 2), 73 DIYTFDGALNKSTVPTDFSSAK 115gi|68223315 (isoform 3) 111 MDKSDAVSHTGNYTCEVTELTR 116 11 CD63 antigengi|4502679 (isoform A), X ◯ X X 130 QQMENYPKNNHTASILDR 117gi|91199546 (isoform B) 130 NNHTASILDR 118 172 NRVPDSCCINVTVGCGINFNEK119 12 CD97 antigen gi|17978491 (isoform 1), X X ◯ X 108TFKNESENTCQDVDECQQNPR 120 gi|17978489 (isoform 2), 453 RLSAVNSIFLSHNNTK121 gi|68508955 (isoform 3) 360 RLSAVNSIFLSHNNTK 122 108TFKNESENTCQDVDECQQNPR 123 404 RLSAVNSIFLSHNNTK 124 13complement factor I gi|119392081 X X ◯ X 103 FLNNGTCTAEGK 125 177FKLSDLSINSTECLHVHCR 126 177 LSDLSINSTECLHVHCR 127 464SIPACVPWSPYLFQPNDTCIVSGWGR 128 494 LISNCSKFYGNR 129 14 cystatin Fgi|20302139 X X ◯ ◯ 84 YSVEKFNNCTNDMFLFK 130 84 FNNCTNDMFLFKESR 131 137LDDCDFQTNHTLK 132 15 desmocollin 2 gi|13435364 X X X ◯ 546SLDREAETIKNGIYNITVLASDQGGR 133 629 AINDTAAR 134 16 epithelial V-likegi|21536337 X X X ◯ 39 VLEAVNGTDAR 135 antigen 1 118 LQFDDNGTYTCQVK 13617 FAT tumor suppressor 1 gi|66346693 X X ◯ ◯ 333 AIGGIDWDSHPFGYNLTLQAK137 998 QVYNLTVR 138 1551 IVVNVSDTNDHAPWFTASSYK 139 3716QLLHKINSSVTDIEEIIGVR 140 18 fibrinogen-like 2 gi|5730075 X X ◯ ◯ 263LDGSTNFTR 141 336 LHVGNYNGTAGDALR 142 19 Fraser syndrome 1 gi|108773804X X ◯ ◯ 1107 IHTPSLHVNGSLILPIGSIKPLDFSLLNVQDQEGR 143 1503IVYNITLPLHPNQGIIEHR 144 1776 ISGSEVEELSEVSNFTMEDINNKK 145 2562 YTSYNVSEK146 2667 VIINDTEDEPTLEFDKK 147 20 growth differentiation gi|4758936 X XX ◯ 70 LRANQSWEDSNTDLVPAPAVR 148 factor 15 70 ANQSWEDSNTDLVPAPAVRILTPEVR149 21 immunoglobulin gi|148664190 (isoform 1), ◯ X X X 101 FQLLNFSSSELK150 superfamily, member 4D gi|148664211 (isoform 2) 113 VSLTNVSISDEGR151 isoform 1 22 integrin, alpha 1 gi|31657142 X X X ◯ 418 NTTFNVESTK152 883 DSCESNHNITCK 153 1113 SENASLVLSSSNQK 154 23intercellular adhesion gi|4557878 X X ◯ X 202TELDLRPQGLELFENTSAPYQLQTFVLPATPPQLVSPR 155 molecule 1 267LNPTVTYGNDSFSAK 156 24 interleukin 6 receptor gi|4504673 X X X ◯ 93SVQLHDSGNYSCYR 157 25 latent transforming growth gi|18497288 X X ◯ X 89DSCQQGSNMTLIGENGHSTDTLTGSGFR 158 factor beta binding 349RLNSTHCQDINECAMPGVCR 159 protein 3 845 DRSHCEDIDECDFPAACIGGDCINTNGSYR160 26 mucin 16 gi|83367077 X X ◯ X 12586 NTSVGLLYSGCR 161 13193KFNITESVLQGLLKPLFK 162 14363 NIEDALNQLFRNSSIK 163 14417 NGTQLQNFTLDR 16427 netrin 4 gi|93204871 X X ◯ X 56 KLWADTTCGQNATELYCFYSENTDLTCRQPK 165163 YFATNCSATFGLEDDVVKK 166 28 neuronal cell adhesion gi|81158226 X ◯ XX 223 FNHTQTIQQK 167 molecule isoform A 245, 251VISVDELNDTIAANLSDTEFYGAK 168 276 ERPPTFLTPEGNASNKEELR 169 314 EDGMLPKNR170 507 GSALHEDIYVLHENGTLEIPVAQKDSTGTYTCVAR 171 858 VNVVNSTLAEVHWDPVPLK172 29 olfactomedin related ER gi|17136143 (isoform 1), ◯ ◯ X X 85QLLEKVQNMSQSIEVLDR 173 localized protein gi|5453547 (isoform 2) 85VQNMSQSIEVLDRR 174 270 SMVDFMNTDNFTSHR 175 376 LDPVSLQTLQTWNTSYPKR 17685 QLLEKVQNMSQSIEVLDR 177 30 osteoprotegerin gi|148743793 X X ◯ ◯ 98ELQYVKQECNR 178 152 CPDGFFSNETSSKAPCR 179 178 GNATHDNICSGNSESTQK 180 289HIGHANLTFEQLR 181 31 palmitoyl-protein gi|4506031 X X X ◯ 212 GINESYKK182 thioesterase 1 (ceroid- 232 FLNDSIVDPVDSEWFGFYR 183lipofuscinosis, neuronal 1, infantile) 32 peptidylprolyl isomerasegi|4758950 X X X ◯ 148 HYGPGWVSMANAGKDTNGSQFFITTVK 184 B 33plasminogen activator, gi|4505861 X X ◯ ◯ 152GTWSTAESGAECTNWNSSALAQKPYSGR 185 tissue type I 219AGKYSSEFCSTPACSEGNSDCYFGNGSAYR 186 219 YSSEFCSTPACSEGNSDCYFGNGSAYR 187483 CTSQHLLNRTVTDNMLCAGDTR 188 34 prion protein gi|122056623, X X X ◯197 QHTVTTTTKGENFTETDVK 189 gi|122056625, 197 GENFTETDVK 190gi|122056628, 35 prostaglandin H2 D- gi|32171249 X X X ◯ 51WFSAGLASNSSWLR 191 isomerase 78 SVVAPATDGGLNLTSTFLR 192 36protein tyrosine gi|109633041 (isoform 1), ◯ X X X 721 KVEVEPLNSTAVHVYWK193 phosphatase, receptor gi|109633039 (isoform 2), 966DINSQQELQNITTDTRFTLTGLKPDTTYDIK 194 type, F 721 KVEVEPLNSTAVHVYWK 195957 DINSQQELQNITTDTRFTLTGLKPDTTYDIK 196 37 protein tyrosinegi|110735404 (isoform 1), X X ◯ X 410 QLTLQWEPLGYNVTR 197phosphatase, receptor gi|110735406 (isoform 2), type, Ugi|l10735402 (isoform 3) 38 seizure related 6 gi|6912612 (isoform 1), ◯◯ X X 177 LLANSSMLGEGQVLR 198 homolog (mouse)-like 2gi|42491358 (isoform 2) 303 IVSPEPGGAVGPNLTCR 199 247 LLANSSMLGEGQVLR200 373 IVSPEPGGAVGPNLTCR 201 39 seizure related 6 gi|32261332 ◯ X X X328 SVNLSDGELLSIR 202 homolog (mouse)-like 40 seizure related 6gi|148839280 (isoform 1), X ◯ X X 399, 422HLTCLNATQPFWDSKEPVCIAACGGVIRNATTGR 203 homolog gi|148839346 (isoform 2)436, 440 IVSPGFPGNYSNNLTCHWLLEAPEGQR 204 41 serine carboxypeptidasegi|83641874, ◯ ◯ X X 346 QAIHVGNQTFNDGTIVEK 205 vitellogenic-likegi|83641876 42 solute carrier family 39 gi|55741750 X X ◯ X 191, 198LHHHLDHNNTHHFHNDSITPSER 206 (zinc transporter), 218GEPSNEPSTETNKTQEQSDVKLPK 207 member 10 339 KDLNEDDHHHECLNVTQLLK 208 43tenascin C (hexabrachion) gi|4504549 X X ◯ X 38 QSGVNATLPEENQPVVFNHVYNIK209 327 CINGTCYCEEGFTGEDCGKPTCPHACHTQGR 210 788QTGLAPGQEYEISLHIVKNNTRGPGLK 211 1018 LNYSLPTGQWVGVQLPR 212 1034NTTSYVLRGLEPGQEYNVLLTAEK 213 1079 VKASTEQAPELENLTVTEVGWDGLR 214 1093LNWTAADQAYEHFIIQVQEANKVEAAR 215 1485 LLETVEYNISGAER 216 44tissue factor pathway gi|5454114 X X ◯ X 145 YFYNNQTK 217 inhibitor 45transforming growth gi|63025222 X X ◯ ◯ 82 LRLASPPSQGEVPPGPLPEAVLALYNSTR218 factor, beta 1 46 tumor-associated calcium gi|4505059, X X ◯ X 111QCNGTSTCWCVNTAGVR 219 signal transducer 1 gi|4505057 168HRPTAGAFNHSDLDAELR 220 47 UDP-GIcNAc:betaGal beta- gi|9845238 X X ◯ X 89LSNISHLNYCEPDLR 221 1,3-N-acetyl- glucosaminyltransferase 2 173ESWGQESNAGNQTVVR 222 48 von Willebrand factor A gi|38348304 X X ◯ ◯ 147NASVPQILIIVTDGK 223 domain containing 2

(2) The lung cancer differential marker glycoprotein according to (1),wherein the sugar chain is at least one glycan selected from the groupconsisting of a fucosylated glycan, a high mannose-type glycan, ahybrid-type glycan, a biantennary complex-type glycan, chitin,polylactosamine, and a β1,3-galactose epitope.

(3) The lung cancer differential marker glycoprotein according to (2),wherein the glycoprotein is for differential diagnosis of small celllung cancer or lung adenocarcinoma.

(4) The lung cancer differential marker glycoprotein according to (3),wherein the glycoprotein is for differential diagnosis of small celllung cancer and is at least one glycoprotein selected from the groupconsisting of neural cell adhesion molecule (NCAM1), secretogranin III,and insulin-like growth factor-binding protein-L1 (IGFBP-L1).

(5) The lung cancer differential marker glycoprotein according to (3),wherein the glycoprotein is for differential diagnosis of lungadenocarcinoma and is fibronectin 1.

(6) A fragment of a lung cancer differential marker glycoproteinaccording to any of (1) to (5), comprising at least one asparagineresidue at a glycosylation site shown in Table 1 or 2 being glycosylatedwith a glycan.

(7) A method for determining lung cancer, comprising detecting at leastone lung cancer differential marker glycoprotein shown in Table 1 or 2being glycosylated with a glycan at the asparagine residue(s) at theglycosylation site(s) shown in Table 1 or 2, and/or at least onefragment thereof, the fragment comprising at least one asparagineresidue at the glycosylation site shown in Table 1 or 2 beingglycosylated with a glycan, from a sample obtained from a test subject,wherein the detection of the glycoprotein or fragment determines thatthe test subject suffers lung cancer.

(8) The method according to (7), wherein the lung cancer differentialmarker glycoprotein and/or the fragment thereof are detected using atleast one glycan probe binding to the glycan.

(9) The method according to (8), wherein the glycan probe binds to afucosylated glycan, a high mannose-type glycan, a hybrid-type glycan, abiantennary complex-type glycan, chitin, polylactosamine, or aβ1,3-galactose epitope.

(10) The method according to (8) or (9), wherein the glycan probe is alectin, an antibody, or a phage antibody.

(11) The method according to (10), wherein the lectin is AAL, ConA, PWM,or PNA.

(12) The method according to (11), wherein the lung cancer differentialmarker glycoprotein is neural cell adhesion molecule (NCAM1), and thedetection of the binding thereof to AAL determines the histological typeof the lung cancer as small cell cancer.

(13) The method according to (11), wherein the lung cancer differentialmarker glycoprotein is secretogranin III, and the detection of thebinding thereof to AAL and/or ConA determines the histological type ofthe lung cancer as small cell cancer.

(14) The method according to (11), wherein the lung cancer differentialmarker glycoprotein is insulin-like growth factor-binding protein-L1(IGFBP-L1), and the detection of the binding thereof to ConA and/or PWMdetermines the histological type of the lung cancer as small cellcancer.

(15) The method according to (11), wherein the lung cancer differentialmarker glycoprotein is fibronectin 1, and the detection of the bindingthereof to AAL and/or PNA determines the histological type of the lungcancer as adenocarcinoma.

(16) The method according to any of (8) to (11), wherein thehistological type of lung cancer is determined as small cell cancer oradenocarcinoma on the basis of a result of binding of the glycan probeto the glycan in the lung cancer differential marker glycoprotein and/orthe fragment thereof, and a manner of binding of the lung cancerdifferential marker glycoprotein shown in Table 1 or 2 and/or thefragment thereof to the glycan probe.

(17) The method according to any of (7) to (16), wherein the sample is abody fluid, a cell, or a lung lavage.

(18) The method according to (17), wherein the body fluid is blood(including serum, plasma, and interstitial fluid), lymph, a cellextract, sputum, or pleural effusion.

(19) A lung cancer cell-identifying antibody for histological staining,binding to a lung cancer differential marker glycoprotein listed inTable 1 or 2 being glycosylated with a glycan at the asparagineresidue(s) at the glycosylation site(s) shown in Table 1 or 2, and/or afragment thereof, the fragment comprising at least one asparagineresidue at a glycosylation site shown in Table 1 or 2 being glycosylatedwith a glycan, and thereby diagnosing lung cancer.

(20) The antibody according to (19), wherein the antibody is capable ofdetermining a histological type of a lung cancer cell.

(21) The antibody according to (20), wherein the lung cancerdifferential marker glycoprotein is neuronal pentraxin receptor (NPR),and the histological type of the lung cancer cell is determined as smallcell cancer.

The present specification encompasses the contents described in thespecification and/or drawings of Japanese Patent Application No.2010-209932 on which the priority of the present application is based.

Advantageous Effects of Invention

According to the lung cancer differential marker of the presentinvention, the presence or absence of lung cancer can be determinedconveniently and highly reliably by the testing of a body fluid, a cell,or a lung lavage. According to the lung cancer differential marker ofthe present invention, the histological type of lung cancer can befurther determined.

The method of the present invention can determine the presence orabsence of lung cancer and the histological type of lung cancer using abody fluid, a cell, or a lung lavage with a more highly properdifferential rate and lower invasiveness than existing tumor markers.

According to the glycan probe for differential diagnosis of lung cancerof the present invention, the presence or absence of lung cancer andfurther, its histological type can be determined by histologicalstaining.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a Western blot image showing the expression of small cell lungcancer differential marker glycoproteins in the culture supernatants ofcultured small cell lung cancer cells (Sc) and cultured lungadenocarcinoma cells (Ad).

FIG. 2 is a Western blot image showing the expression of lungadenocarcinoma differential marker glycoproteins in the culturesupernatants of cultured small cell lung cancer cells (Sc) and culturedlung adenocarcinoma cells (Ad).

FIG. 3 is a Western blot image in which lung cancer differential markerglycoproteins in the culture supernatants of cultured small cell lungcancer cells (Sc) and cultured lung adenocarcinoma cells (Ad)fractionated with each lectin were detected with their respectiveanti-lung cancer differential marker protein antibodies.

FIG. 4 is a blot image in which a lung cancer differential markerglycoprotein (fibronectin 1) in the culture supernatants of culturedsmall cell lung cancer cells (Sc) and cultured lung adenocarcinoma cells(Ad) fractionated with an antibody (anti-fibronectin 1 antibody) wasdetected with AAL lectin.

FIG. 5 is a Western blot image in which lung cancer differential markerglycoproteins (NCAM and fibronectin 1) in the sera of small cell lungcancer patients (Sc) and lung adenocarcinoma patients (Ad) fractionatedwith AAL lectin were detected with their respective anti-lung cancerdifferential marker glycoprotein antibodies

FIG. 6 is a Western blot image in which a lung cancer differentialmarker glycoprotein (secretogranin III) in the sera of small cell lungcancer patients (Sc) and lung adenocarcinoma patients (Ad) fractionatedby the serial chromatography of the sera was detected with an antibody(anti-secretogranin III antibody).

FIG. 7 is an image of histological staining of each histological type oflung cancer with an anti-neuronal pentraxin receptor (NPR) antibody.

FIG. 8 is a Western blot image in which a lung cancer differentialmarker glycoprotein (secretogranin III) in the sera of small cell lungcancer patients (Sc) and lung adenocarcinoma patients (Ad) fractionatedby the multisample serial chromatography of the sera was detected withan antibody (anti-secretogranin III antibody).

FIG. 9 is a diagram showing the comparison of fluorescence signalintensities on PWM lectin spots, from a lung cancer differential markerglycoprotein (insulin-like growth factor-binding protein-L1), in anantibody-overlay lectin array using eluates purified using an antibody(anti-insulin-like growth factor-binding protein-L1 antibody) from theculture supernatants of cultured small cell lung cancer cells (Sc) andcultured lung adenocarcinoma cells (Ad).

FIG. 10 is a diagram showing the comparison of fluorescence signalintensities on PNA lectin spots, from a lung cancer differential markerglycoprotein (fibronectin 1), in an antibody-overlay lectin array usingeluates purified using an antibody (anti-fibronectin 1 antibody) fromthe culture supernatants of cultured small cell lung cancer cells (Sc)and cultured lung adenocarcinoma cells (Ad).

DESCRIPTION OF EMBODIMENTS 1. Lung Cancer Differential MarkerGlycoprotein and Fragment Thereof

The first embodiment of the present invention provides lung cancerdifferential marker glycoproteins shown in Table 1 or 2 above andfragment thereof.

1-1. Lung Cancer Differential Marker Glycoprotein

The “lung cancer differential marker glycoprotein” of this embodimentcorresponds to a protein represented by each of Protein #1 to #31 inTable 1 and Protein #1 to #48 in Table 2. All of these proteins are lungcancer-specific glycoproteins comprising, in their amino acid sequences,a glycosylated asparagine residue at least at a position (counted fromthe initiating amino acid residue as the first position) represented by“Glycosylation site” in each table. In the case of, for example, acidalpha-glucosidase represented by Protein #1 in Table 1, glycans arelinked to asparagine residues at least at positions 390, 470, and 882 inthe amino acid sequence of this protein. Hereinafter, in the presentspecification, such a glycosylated protein is referred to as a“glycoprotein”.

In each table, “gi(ID)” represents the ID number of each glycoprotein ofthis embodiment. A plurality of gi(ID) numbers registered for oneprotein are all described in the table. Also, a plurality of isoforms ofone protein are indicated by isoform numbers together with their gi(ID)numbers in the table.

The glycan linked to the asparagine residue is not particularly limitedas long as the glycan is linked in a lung cancer-specific manner.Examples thereof include fucosylated glycans, high mannose-type glycans,hybrid-type glycans, biantennary complex-type glycans, chitin,polylactosamine, and β1,3-galactose epitopes. In this context, the“glycan linked in a lung cancer-specific manner” refers to a glycanlinked to the asparagine residue represented by “Glycosylation site” inthe table, only in the lung cancer cell-derived protein. Hence, as arule, the lung cancer differential marker glycoprotein of thisembodiment is a glycoprotein produced from a lung cancer cell. Thus, thedetection of presence or absence of the glycoprotein, for example, inthe serum of a test subject, using a glycan probe recognizing thisglycoprotein can determine an individual having the glycoprotein assuffering lung cancer.

In the present specification, the “glycan probe” refers to a determinantthat specifically recognizes a particular glycan and/or glycoconjugatesuch as a glycoprotein and binds thereto. Examples thereof includelectins, antibodies, and phage antibodies.

As described above, the histological types of “lung cancer” are known toconsist of: small cell cancer; and non-small cell cancer furtherincluding adenocarcinoma, squamous cell carcinoma, and large cellcancer. Neuroendocrine cancer in the lung is also known. Most of theneuroendocrine cancer types are classified into small cell cancer,whereas the other histological types of this cancer are also known. Thelung cancer differential marker glycoprotein of this embodiment candetermine the histological type of lung cancer, depending on the type ofthe glycan linked to the prescribed asparagine residue. In the case of,for example, acid alpha-glucosidase represented by Protein #1 in Table1, fucosylated glycans are linked to the prescribed asparagine residuesonly in a small cell lung cancer-derived glycoprotein. Thus, use of alectin or an antibody that can bind to and recognize any of thefucosylated glycans can diagnose lung cancer with acid alpha-glucosidaseas a lung cancer differential marker glycoprotein. In this case, theacid alpha-glucosidase can also serve as a marker that confirms the lungcancer as small cell cancer. Alternatively, in the case of biotimidaserepresented by Protein #2 in Table 1, fucosylated glycans are linked tothe prescribed asparagine residues only in a lung adenocarcinoma-derivedglycoprotein. Thus, use of a lectin or an antibody that can bind to andrecognize any of the fucosylated glycans can diagnose lung cancer withbiotimidase as a lung cancer differential marker glycoprotein. In thiscase, the biotimidase can also serve as a marker that confirms the lungcancer as adenocarcinoma.

The fucosylated glycan can be detected using AAL lectin. The highmannose-type glycan, the hybrid-type glycan, and the biantennarycomplex-type glycan can be detected using ConA lectin. The chitin andthe polylactosamine can be detected using PWM lectin. The β1,3-galactoseepitope can be detected using PNA lectin. In Table 1, the presence andabsence of the binding between each lung cancer differential markerglycoprotein and AAL lectin or ConA lectin are indicated by “◯” and “x”,respectively. Taking acid alpha-glucosidase represented by Protein #1 asan example, only a small cell lung cancer-derived glycoprotein hasfucosylated glycans at the prescribed asparagine residues and is thusindicated by “◯” in “AAL” of “Small cell cancer” and by “x” in the otherboxes. The same holds true for the other lung cancer differential markerglycoproteins in Tables 1 and 2. Accordingly, each lung cancerdifferential marker glycoprotein (including a fragment of the lungcancer differential marker glycoprotein) shown in Tables 1 and 2 canserve as a marker for differentiation of small cell lung cancer or lungadenocarcinoma based on the manner of its binding to AAL lectin and ConAlectin for “small cell cancer” and “adenocarcinoma” in Tables 1 and 2.

1-2. Fragment of Lung Cancer Differential Marker Glycoprotein

The “lung cancer differential marker glycoprotein fragment” or the“fragment of the lung cancer differential marker glycoprotein” of thisembodiment refers to an oligopeptide or polypeptide fragment consistingof a portion of the lung cancer differential marker glycoprotein. Thisfragment comprises, in its amino acid sequence, at least one asparagineresidue at the glycosylation site shown in Table 1 or 2, wherein thelung cancer-specific glycan described in the paragraph “1-1. Lung cancerdifferential marker glycoprotein” is linked to this asparagine residue.

The amino acid length of the lung cancer differential markerglycoprotein fragment is not particularly limited and is preferably 5 to100 amino acids, 8 to 80 amino acids, or 8 to 50 amino acids.Hereinafter, in the present specification, the lung cancer differentialmarker glycoprotein fragment is also referred to as a “glycoproteinfragment” or a “glycopeptide”. Hereinafter, the lung cancer differentialmarker glycoprotein and its glycoprotein fragment are also collectivelyreferred to as a “lung cancer differential marker”.

Specific examples of the lung cancer differential marker glycoproteinfragment include glycopeptides consisting of amino acid sequencesrepresented by SEQ ID NOs: 1 to 223 shown in Tables 1 and 2. These areglycoprotein fragments that were obtained by an IGOT method (describedlater) in identifying the lung cancer differential marker glycoproteinsof this embodiment. Any of these glycoprotein fragments can be used as alung cancer differential marker, as in the lung cancer differentialmarker glycoproteins, to determine the presence or absence of lungcancer and determine the histological type of the detected lung cancer,depending on the type of glycan linked to the prescribed asparagineresidue. In each amino acid sequence shown in Tables 1 and 2, theunderlined asparagine residue (N) represents a glycan-linked asparagineresidue.

1-3. Obtainment of Lung Cancer Diagnosis Marker Glycoprotein

Methods by which the lung cancer differential marker glycoproteinfragments and the lung cancer differential marker glycoproteins of thisembodiment were obtained and identified will be described below.

1-3-1. Large-Scale Identification of Lung Cancer-Specific CandidateGlycoprotein

The large-scale selective collection and concentration of the lungcancer-specific glycoproteins can adopt any of methods broadlyclassified into, for example, a method using probes having affinity forglycans, a method using chemical reaction with glycans (Zhang H. et al.,Nat Biotechnol 21, 660-666 (2003)), and a method of introducing affinitytags to glycans. Here, the method using probes, which were used in theobtainment of the lung cancer differential marker glycoproteins inTables 1 and 2, will be described.

First, lectins or anti-glycan antibodies reactive with glycanscharacteristically produced by cancer cells are selected as probes.

The probe lectins can be selected by the statistical analysis of glycanprofiles using a lectin microarray. Alternatively, the probe lectins maybe selected in consideration of literature information (some probelectins to be used can be expected on the basis of, for example,increased fucosylation associated with malignant transformation) or theresulting determination performance of the histological type. Basically,lectins are selected by the statistical analysis of profile, and theselected lectins are validated on the basis of binding specificity. Inthe case of targeting lung cancer, for example, Aleuria aurantia-derivedAAL lectin capable of detecting fucosylation, Canavaliaensiformis-derived ConA lectin capable of detecting high mannose-type,hybrid-type, or biantennary complex-type glycans, Phytolaccaamericana-derived PWM lectin capable of detecting chitin orpolylactosamine, and/or Arachis hypogaea-derived PNA lectin capable ofdetecting β1,3-galactose epitopes can be used.

The antibody probes or phage antibody probes may be prepared afterstructural determination of antigens (glycans). This is not a necessarycondition, and these probes can also be prepared against the unknownstructures of the antigenic glycans or glycopeptides.

For the large-scale identification of candidate glycoproteins,specifically, candidate glycoproteins are first collected from theculture supernatant of a lung cancer-derived cell line in a medium usingthe glycan probes (probe lectins and/or antibody probes or phageantibody probes). Use of the lung cancer-derived cell culturesupernatant enables cancer cell-derived glycoproteins to be identifiedand can thus facilitate the detection of difference from glycans presenton the serum-derived glycoproteins of healthy persons. This informationcan be useful in selectively obtaining candidate molecules from serum.The cells used may be derived from any histological type of lung cancer,i.e., small cell cancer or non-small cell cancer (adenocarcinoma,squamous cell carcinoma, or large cell cancer). Candidate glycoproteinsmay be collected from each histological type, and the results can beanalyzed by comparison to obtain histological type-nonspecific lungcancer differential markers or histological type-specific lung cancerdifferential markers.

1-3-2. Identification of Glycopeptide by IGOT Method

The collected lung cancer differential marker candidate glycoproteinsare then treated by a method based on an isotope-coded glycosylationsite-specific tagging (IGOT) method and mass spectrometry to identifycore glycopeptides in the candidate glycoproteins. The glycopeptides canbe analyzed by a Lec-IGOT-LC/MS method described in, for example, JPPatent Publication (Kokai) No. 2004-233303 A (2004) (JP Patent No.4220257) and Kaji H, et al., Mass spectrometric identification ofN-linked glycopeptides using lectin-mediated affinity capture andglycosylation site-specific stable isotope tagging. Nature Protocols 1,3019-3027 (2006). An example of the specific method will be describedbelow.

(1) Glycan Cleavage and IGOT Method

The candidate glycoprotein groups collected with the probe lectins orthe probe antibodies are digested with protease. From the obtainedpeptide groups, candidate glycopeptide groups are sampled andre-collected using the same probes as above. Alternatively, crude sampleprotein mixtures may be digested with protease without separatingcandidate glycoprotein groups from the crude sample protein mixtures,and candidate glycopeptide groups can be collected directly from theobtained crude peptide groups using the probe lectins or the probeantibodies. Subsequently, the obtained candidate glycopeptides arelabeled by the IGOT method. The IGOT method involves dissociatingglycans from the glycopeptides by treatment with enzymes such asglycopeptidase in oxygen isotope-labeled water, while causing theconversion of glycosylated asparagine to aspartic acid, during which theoxygen isotope (¹⁸O) in the water is incorporated into the candidateglycopeptides.

(2) LC/MS Shotgun Analysis of Labeled Peptide

The candidate glycopeptides labeled by the IGOT method are separated byliquid column chromatography (LC) and applied to mass spectrometry (MS).Their amino acid sequences are exhaustively identified by tandem massspectrometry.

(3) Identification of Candidate Glycopeptide

According to, for example, an MS/MS ion search method described in theStandard Technology Collection (edited by the Japan Patent Office), Massspectrometry, 3-6-2-2 Amino acid sequence analysis, a database can besearched by comparing the respective MS/MS peptide measurement resultsof the obtained candidate glycopeptides with the hypothetical MS/MSspectra of all peptides predicted according to the specificity of theprotease actually used from the amino acid sequences of all proteinsregistered in the database. In this search, the following amino acidmodifications are taken into consideration: oxidation of methionineresidue side chain, pyroglutamic acid conversion (deamination orcyclization) of ammo-terminal glutamine, deamination of an aminoterminus (carbamidomethylation of cysteine), deamidation of asparagineresidue side chain (provided that the stable isotope oxygen is alreadyincorporated therein). The candidate glycoproteins including thecandidate glycopeptide are identified using search software Mascot.

(4) Identification of Glycosylation Site

Of the candidate glycopeptides identified by the MS/MS ion searchmethod, each candidate glycopeptide that has an asparagine residue withdeamidated (stable isotope-incorporated) side chain and contains anN-linked glycosylation consensus sequence (Asn-Xaa-[Ser/Thr], providedthat Xaa is not Pro) is regarded as the glycoprotein fragment of thisembodiment. Also, the asparagine residue in the consensus sequence ofthe obtained glycoprotein fragment is defined as the glycosylation site.When Xaa is Lys/Arg and the identified peptide sequence is cleaved atthis site, the candidate glycopeptide in which a residue subsequent toXaa is [Ser/Thr] with reference to the amino acid sequence of the wholeprotein thereof is also included in the glycoprotein fragment. As arule, the glycosylation site is contained in the consensus sequence,although some reports suggest different glycosylation sites. In thisspecification, a glycosylation site cleaved with peptide-N-glycanase(glycopeptidase F, PNGase) can be identified.

The lung cancer differential marker glycoprotein fragment groupsselected by the method described above and the lung cancer differentialmarker glycoproteins (comprising these fragments) identified on thebasis of their amino acid sequences are defined as the glycoproteinfragment groups and the glycoproteins shown in Tables 1 and 2.

The glycoprotein fragment groups and the glycoproteins can serve as lungcancer differential markers for diagnosing lung cancer in a testsubject, for example, by testing the presence or absence thereof in theserum of the test subject using lectins or antibodies recognizing them.

1-3-3. Validation of Lung Cancer Differential Marker

The significance of the lung cancer differential markers selected in theparagraph 1-3-2 can be further validated. For example, from among theglycoproteins and/or the glycoprotein fragments collected with the probelectins from the lung cancer cell culture supernatant, significant lungcancer differential markers can be detected and confirmed by Westernblot or immunoprecipitation using antibodies specifically recognizingthe lung cancer differential markers separated and identified above. Inthe present specification, 31 lung cancer differential markerglycoproteins shown in Table 1 and fragments of the glycoproteinscorrespond to the lung cancer differential markers further selected bythe detection with the antibodies.

Also, the lung cancer differential markers further selected by thedetection with the antibodies may be further validated. For example,lung cancer differential markers are collected using the probe lectinsfrom the sera of lung cancer patients, and significant markers can befurther detected using the specific antibodies. As an example, neuralcell adhesion molecule (NCAM 1) (represented by Protein #19 in Table 1)and fibronectin 1 (represented by Protein #8 in Table 1) shown in FIG.5, and secretogranin III (represented by Protein #23 in Table 1) shownin FIGS. 6 and 8 correspond to the lung cancer differential markerglycoproteins confirmed to be useful by this further validation. In thiscontext, the neural cell adhesion molecule binds to AAL lectin only insmall cell lung cancer as shown in Table 1 and can thus serve as a lungcancer differential marker that allows differential diagnosis of smallcell lung cancer. Likewise, the secretogranin III binds to AAL lectinand ConA lectin only in small cell lung cancer as shown in Table 1 andcan thus serve as a lung cancer differential marker that allowsdifferential diagnosis of small cell lung cancer. By contrast, thefibronectin 1 binds to AAL lectin only in lung adenocarcinoma as shownin Table 1 and can thus serve as a lung cancer differential marker thatallows differential diagnosis of lung adenocarcinoma. Alternatively, forexample, lung cancer differential markers may be collected from theculture supernatant of a lung cancer cell line using specificantibodies, and useful markers can be detected using a lectin array. Asan example, insulin-like growth factor-binding protein-L1 (IGFBP-L1)(represented by Protein #12 in Table 1) shown in FIG. 9 and fibronectin1 (represented by Protein #8 in Table 1) shown in FIG. 10 correspond tothe lung cancer differential marker glycoproteins confirmed to be usefulby the further validation. In this context, the insulin-like growthfactor-binding protein-L1 (IGFBP-L1) produces a strong detectablefluorescence signal on a PWM lectin spot in small cell lung cancer asshown in FIG. 9 and can thus serve as a lung cancer differential markerthat allows differential diagnosis of small cell lung cancer. Bycontrast, the fibronectin 1 produces a detectable fluorescence signal ona PNA lectin spot only in lung adenocarcinoma as shown in FIG. 10 andcan thus serve as a lung cancer differential marker that allowsdifferential diagnosis of lung adenocarcinoma.

1-4. Detection of Lung Cancer Differential Marker Glycopeptide andGlycoprotein 1-4-1. Mass Spectrometry

The lung cancer differential marker glycopeptides and glycoproteins canbe detected with a mass spectrometer as a detector from samplescollected with the probe lectins or the like binding to glycans.

The collected lung cancer differential marker glycopeptides can bedetected, preferably, by the cleavage of their glycans followed byliquid chromatography (LC) to separate and elute peptides, which arethen introduced in order directly to a mass spectrometer (MS) on line.The mass spectrometry is not only available for obtaining mass spectra,but also available for obtaining MS/MS spectra using a fragmentationmethod such as collision-induced dissociation (CID) and further detect aplurality of fragment ions generated by CID or the like only whenpre-selected ions are detected (this approach is also called singlereaction monitoring or multiple reaction monitoring). Furthermore,analyte peptides differing in mass by the incorporation of stableisotopes into some of synthesized core peptide moieties of lung cancerdifferential marker glycopeptides may be added to analysis samples, andtheir respective signal intensities can be compared to thereby achieverelative or absolute quantitative analysis. More simply, the signalintensity of the detected ion may be compared between two or moresamples or with that of a reference sample to achieve simplifiedquantification.

The lung cancer differential marker glycoproteins can be detected usingvarious proteomics approaches known in the art. For example, thecollected proteins can be separated by one-dimensional ortwo-dimensional gel electrophoresis and quantified by the comparison ofsignal intensities (dye, fluorescence, etc.) between the target spot anda reference sample. In the case of a detection method using a massspectrometer, the collected protein groups are digested with protease,and the formed peptides can be detected by LC/MS analysis. For suchquantification, various methods based on stable isotope labeling (ICAT,MCAT, iTRAQ, SILAC, etc.) can be used in combination with a non-labeledsimple quantification method (peptide counting method, area integrationmethod, etc.). As further described later, the glycoproteins may bequantified by ELISA.

1-4-2. Lectin Microarray

(1) Glycan Profiling Using Lectin Microarray

(a) Lectin Microarray (Also Simply Referred to as a Lectin Array)

The lectin array refers to a substrate on which plural types of lectinsdiffering in specificity are immobilized in parallel (i.e., in the formof array) as glycan probes. The lectin array allows concurrent analysison the types of lectins interacted with analyte glycoconjugates and thedegrees of these interactions. By use of this lectin array, informationrequired for estimating glycan structures can be obtained by singleanalysis, while the steps from sample preparation to scanning can becarried out quickly and conveniently. In a glycan profiling system suchas mass spectrometry, glycoproteins cannot be analyzed directly and mustthus be decomposed into glycopeptides or free glycans in advance. In thelectin microarray, advantageously, glycoproteins can be analyzeddirectly by merely introducing, for example, fluorescent materialsdirectly into the core protein moieties thereof. The lectin microarraytechnique has been developed by the present inventors, and thefundamental principles of this technique are described in, for example,Kuno A., et al. Nat. Methods 2, 851-856 (2005).

Typical examples of the lectins used in the lectin array include thosedescribed in the following Table 3:

[Table 3] #1 Sugar Binding Specificity

For example, a lectin array comprising 45 types of lectins (includingthe 43 types of lectins described above) immobilized on a substrate isalready commercially available under the product name of LecChip from GPBiosciences Ltd.

(2) Statistical Analysis of Glycan Profiles Using Lectin Array

The lectin array has already evolved into a practical technique by whicha quantitative comparative glycan profiling can be realized not only forpurified preparations but also for mixed samples such as serum or celllysates. Particularly, the comparative glycan profiling of cell surfaceglycans has achieved remarkable development (Ebe, Y et al. J. Biochem.139, 323-327 (2006); Pilobello, K. T. et al. Proc Natl Acad Sci USA.104, 11534-11539 (2007); and Tateno, H. et al. Glycobiology 17,1138-1146 (2007)).

Also, data mining by the statistical analysis of glycan profiles can becarried out by a method shown in, for example, “Kuno A, et al. JProteomics Bioinform. 1. 68-72 (2008)” or “the Japanese Society ofCarbohydrate Research 2008/8/18, Development of Application Techniquefor Lectin Microarray—Comparative Glycan Profiling and StatisticalAnalysis of Biological Sample-, Atsushi Kuno, Atsushi Matsuda, YokoItakura, Hideki Matsuzaki, Hisashi Narimatsu, Jun Hirabayashi” and“Matsuda A, et al. Biochem Biophys Res Commun. 370, 259-263 (2008)”.

(3) Antibody-Overlay Lectin Microarray Method

The platform of the lectin microarray in this method is basically thesame as above. For detection, the sample of the test subject is notdirectly labeled with a fluorescent material or the like but isindirectly labeled by the introduction of a fluorescent group or thelike into the sample of the test subject via an antibody. Thisapplication method can realize concurrent multisample analysis moreconveniently and quickly (see “Kuno A, Kato Y, Matsuda A, Kaneko M K,Ito H, Amano K, Chiba Y, Narimatsu H, Hirabayashi, J. Mol. CellProteomics. 8, 99-108 (2009)”, “Jun Hirabayashi, Atsushi Kuno, NoboruUchiyama, “Development of Application Technology for Glycan ProfilingUsing Lectin Microarray”, Experimental Medicine, extra number “Study onCancer Diagnosis at Molecular Level—Challenge to Clinical Application”,Yodosha Co., Ltd., Vol. 25 (17) 164-171 (2007)”, Atsushi Kuno, JunHirabayashi, “Application of Glycan Profiling System Based On LectinMicroarray to Glycan Biomarker Search”, and Genetic Medicine MOOK No. 11“Development of Clinical Glycan Biomarker and Elucidation of GlycanFunction”, pp. 34-39, Medical Do, Inc. (2008)).

For example, glycan moieties in glycoprotein samples obtained from atest subject are recognized by lectins on the lectin microarray. Thus,antibodies against the core protein moieties thereof can be overlaid onthe glycoproteins to thereby specifically and highly sensitively detectthe glycoproteins without labeling the test glycoproteins or highlypurifying them.

(4) Lectin-Overlay Antibody Microarray Method

This method employs, instead of the lectin microarray, an antibodymicroarray in which antibodies against core proteins are immobilized inparallel (i.e., in the form of array) on a substrate such as a glasssubstrate. This method requires the same numbers of antibodies as thenumber of markers to be examined and also requires determining lectinsin advance for detecting the alteration of glycans.

1-4-3 Lectin-Antibody Sandwich Immunological Detection

A simple and inexpensive sandwich detection method can be designed onthe basis of the results obtained using the lectin array. Basically,this method can adopt the protocol of the sandwich detection methodusing two types of antibodies except that one of the antibodies isreplaced by lectins. Thus, this approach is also applicable toautomatization using an existing automatic immunodetection apparatus.The point to be noted is only the reaction between antibodies andlectins to be used for sandwiching antigens. Each antibody has at leasttwo N-linked glycans. When the lectins used recognize glycans on theantibodies, background noise inevitably occurs during sandwich detectiondue to the binding reaction therebetween. A possible approach forpreventing the generation of this noise signal involves modifying theglycan moieties on the antibodies or using only Fab fragments, whichcontain no such glycan moieties. These approaches have already beenknown in the art. For the method of modifying the glycan moieties, forexample, Chen S. et al., Nat. Methods. 4, 437-44 (2007) and Comunale MA, et al., J Proteome Res. 8, 595-602 (2009) can be referred to. For themethod using Fab fragments, for example, Matsumoto H., et al., Clin ChemLab Med 48, 505-512 (2010) can be referred to.

1-4-4. Method Using Serial Chromatography

The antibody-overlay lectin array is the best approach for statisticallyfinding lectins that most precisely reflect the disease-specificalteration of glycans on the lung cancer differential markerglycoproteins. This approach, however, inevitably requires antibodiesthat permit immunoprecipitation and overlay detection. Nevertheless,such antibodies are not always available. In this regard, theimmunological quantitative detection of target glycoproteins from amongglycoproteins collected with probe lectins is generally carried out asmeans for using a larger number of candidate molecules in the detectionof lung cancer. Specifically, SDS-PAGE is performed, and targetglycoproteins are immunologically detected by Western blot aftermembrane transfer. The signal intensities of the obtained bands can becompared to thereby quantitatively estimate the change between samples.The significance of each marker candidate can be validated on the basisof change in the amount of the protein having the cancer-specificalteration of a glycan, to thereby narrow down the candidateglycoproteins. In this embodiment, generally, the AAL lectin used in thestep of identifying candidate molecules is also used as a probe proteinin this validation. Examples of practice under such a strategy includethe report of Liu Y et al., J Proteome Res. 9, 798-805 (2010). Serumproteins are known to differ in the structures (the degree of branching,etc.) or fucosylation (core fucose, blood group antigen, etc.) of theirN-linked glycans, depending on the types thereof. Reportedly, even thesame molecules may therefore be differently fucosylated. For example,Nakagawa T. et al., J. Biol. Chem. 281, 29797-29806 (2006) disclosesthat al antitrypsin molecules are differently fucosylated. Such proteinsmay or may not be increased at different times depending on the type ofdisease and the stage of its progression. Thus, the approach is notideal, in which all fucose-containing glycoproteins are collected usingAAL probes, which can recognize almost all fucosylation patterns andcollect the glycoproteins, and the collected glycoproteins arequantitatively compared. Hence, we have conceived that proteins areseparated and fractionated by serial chromatography using two differentfucose-recognizing lectins and these fractions are quantitativelycompared and analyzed. The lectins used in this approach are LCA andAAL. Previous lectin specificity analysis has revealed that the LCAlectin recognizes core-fucosylated glycans with fewer branches, amongN-linked glycans. The AAL lectin is known to be capable of recognizingevery core fucose with any number of branches in N-linked glycans aswell as even fucosylation at the non-reducing end typified by ABO, Lewisantigens, or the like. This means that LCA has high specificity whileAAL has low specificity. Thus, in the first step, fucose-containingglycoproteins binding to LCA are captured by LCA column chromatography.These glycoproteins are regarded as LCA-bound fucose-containingglycoproteins. In this chromatography, fucose-containing glycoproteinshaving no core-fucosylated N-linked glycan with fewer branches arefractionated into unbound fractions without binding to the LCA column.In order to capture such fucose-containing glycoprotein groups from theLCA-unbound fractions, the LCA-unbound fractions are subjected to AALcolumn chromatography. The glycoprotein groups captured by AAL in thisstep are regarded as LCA-unbound/AAL-bound fucose-containingglycoproteins. These procedures can presumably evaluate increase ordecrease in fucosylation of the same protein associated with thedisease, on the basis of the type of modification.

2. Method for Determining Lung Cancer

The second embodiment of the present invention provides a method fordetermining lung cancer. The method of this embodiment comprisesdetecting the lung cancer differential marker of embodiment 1 from asample obtained from a test subject, wherein the detection of theglycoprotein or fragment determines that the test subject suffers lungcancer.

2-1. Definition and Summary

In the present specification, the “test subject” refers to a person tobe subjected to a test, i.e., a human donor of a sample described later.The test subject may be any of patients having a certain disease orhealthy persons. The test subject is preferably a person possibly havinglung cancer or a lung cancer patient.

The “sample” refers to a part that is obtained from the test subject andsubjected to the differential method of this embodiment. For example, abody fluid, a cell from cancer tissue or the like, or a lung lavageobtained during operation applies to the sample.

The “body fluid” refers to a biological sample in a liquid stateobtained from the test subject. Examples thereof include blood(including serum, plasma, and interstitial fluid), lymph, extracts ofeach tissue or cell, pleural effusion, sputum, spinal fluid, lacrimalfluid, nasal discharge, saliva, urine, vaginal fluid, and seminal fluid.The body fluid is preferably blood, lymph, pleural effusion, or sputum.The body fluid may be used, if necessary, after treatment such as thedilution or concentration of the sample obtained from the test subjector the addition of an anticoagulant such as heparin thereto.Alternatively, the body fluid may be used directly without suchpretreatment. The body fluid can be obtained by a method known in theart. For example, blood or lymph can be obtained according to a bloodcollection method known in the art. Specifically, peripheral blood canbe obtained from the vein or the like of a peripheral site by injection.The body fluid may be used immediately after obtainment. Alternatively,the body fluid may be cryopreserved or refrigerated for a given periodand then treated, if necessary, by thawing or the like before use. Inthis embodiment, in the case of using serum, sufficient amounts of lungcancer differential markers can be detected using a volume of 10 μL, to100 μL, 20 μL, to 80 μL, to 70 μL to 60 μL, or 45 μL to 55 μL.

In the differential method of this embodiment, the lung cancer diagnosismarker used in detection can be any lung cancer diagnosis marker as longas the marker is a lung cancer marker glycoprotein listed in Table 1 or2 being glycosylated with a glycan at the asparagine residue(s) at theglycosylation site(s) shown in Table 1 or 2, or a glycoprotein fragmentthereof comprising at least one asparagine residue at the glycosylationsite shown in Table 1 or 2 being glycosylated with a glycan. These lungcancer diagnosis markers may be used alone or in combination of two ormore thereof in the diagnosis method of this embodiment. For example,two or more different lung cancer diagnosis marker glycoproteins may beused. Alternatively, two or more different fragments of the same lungcancer diagnosis marker glycoprotein may be used. Preferably, the lungcancer diagnosis marker(s) shown in Table 1 is used. More preferably,lung cancer diagnosis markers having the same or different diagnosticcharacteristics for small cell lung cancer and lung adenocarcinoma areused. Examples of the use method include a method using neural celladhesion molecule (NCAM1), secretogranin III, and/or insulin-like growthfactor-binding protein-L1 (IGFBP-L1) shown in Table 1 for differentialdiagnosis of small cell lung cancer, in combination with fibronectin 1shown in Table 1 for differential diagnosis of lung adenocarcinoma asthe lung cancer differential markers of this embodiment. This method isconvenient because it can determine the presence or absence of lungcancer in the test subject while also determining the histological type(small cell cancer or adenocarcinoma) of the lung cancer. When the lungcancer differential marker shown in Table 1 or 2 is detected from thesample of the test subject by any method using, alone or in combination,lectins or antibodies binding to a fucosylated glycan, a highmannose-type glycan, a hybrid-type glycan, a biantennary complex-typeglycan, chitin, polylactosamine, and/or a β1,3-galactose epitope, thistest subject can be confirmed to have lung cancer or to be highly likelyto have lung cancer. For example, the fucosylated glycan can be detectedusing AAL lectin specifically binding thereto. The high mannose-typeglycan, the hybrid-type glycan, and the biantennary complex-type glycancan be detected using ConA lectin specifically binding thereto. Thechitin and the polylactosamine can be detected using PWM lectinspecifically binding thereto. The β1,3-galactose epitope can be detectedusing PNA lectin specifically binding thereto.

Alternatively, the histological type of lung cancer in the test subjectmay be confirmed as small cell cancer or adenocarcinoma on the basis ofresults of binding of the lectin to the glycan in the lung cancerdifferential marker in the sample obtained from the test subject, andthe manner of binding of the lung cancer differential markerglycoprotein shown in Table 1 or 2 and/or the fragment thereof to thelectin. According to the manner of lectin binding of the glycoproteinshown in Table 1, for example, the glycans of acid alpha-glucosidaserepresented by Protein #1 bind to AAL lectin only in small cell lungcancer. Thus, when acid alpha-glucosidase bound to AAL lectin isdetected from the sample obtained from a test subject, this test subjectcan be confirmed to have lung cancer whose histological type is smallcell cancer. Alternatively, when neural cell adhesion molecule (NCAM1)as the lung cancer differential marker glycoprotein or its glycoproteinfragment bound with AAL lectin is detected, the histological type oflung cancer can be confirmed as small cell cancer. When fibronectin 1 asthe lung cancer differential marker glycoprotein or its glycoproteinfragment bound with AAL lectin or an anti-fibronectin 1 (fragment)antibody is detected, the histological type of lung cancer can beconfirmed as adenocarcinoma.

2-2. Method for Detecting Lung Cancer Differential Marker

Examples of the method for detecting the lung cancer differential markercan include the combination of two methods: a method involving using alectin specifically binding to a glycan in each lung cancer differentialmarker (hereinafter, in the present specification, this lectin isreferred to as lectin A for the sake of convenience) to select a lungcancer differential marker having the glycan; and a method involvingdetecting a moiety (core protein) other than the glycan of each lungcancer differential marker to thereby detect the lung cancerdifferential marker. Alternative examples of the method can include amethod involving using an antibody to detect the lung cancerdifferential marker of interest, the antibody being specific for thelung cancer differential marker having a glycan specifically binding tolectin A and recognizing an epitope located in proximity to theglycosylation site.

In this context, the method of detecting a glycan specifically bindingto lectin A and the method of detecting a core protein may be a methodof assaying a glycan specifically binding to lectin A and a method ofassaying a core protein, respectively. For example, the lung cancerdifferential marker can be detected using an antibody against the coreprotein and lectin A to thereby differentiate between lung cancerpatients and healthy persons. Preferably, the antibody overlay methodusing a lectin array (Kuno A, Kato Y, Matsuda A, Kaneko M K, Ito H,Amano K, Chiba Y, Narimatsu H, Hirabayashi J. Mol. Cell Proteomics. 8,99-108 (2009)) can be used. More simply, the lectin-antibody sandwichimmunological detection method can be used for detection.

Examples of the specific method for determining lung cancer using thelung cancer differential marker having a glycan specifically binding tolectin A include, a method comprising the following steps:

(1) separating lung cancer differential markers using lectin A fromserum obtained from a test subject to thereby select a protein grouphaving the glycan specifically binding to lectin A; and

(2) subsequently screening the lung cancer differential markers using ananti-lung cancer differential marker glycoprotein antibody specificallyrecognizing the moiety other than the glycan specifically binding tolectin A, to thereby detect the lung cancer differential marker ofinterest having the glycan specifically binding to lectin A, whereinwhen this marker is detected, this test subject is determined to beafflicted with lung cancer.

The lung cancer differential marker having the glycan specificallyreacting with lectin A can be selected using, for example, the method ofassaying a glycan specifically binding to lectin A using, specifically,a lectin A-immobilized column or array, and lung cancer differentialmarker assaying means, specifically, an antibody against the novel lungcancer differential marker glycoprotein or the fragment thereof.Preferably, the lectin-antibody sandwich ELISA or the antibody-overlaylectin array method can be used.

Also, the concentration of the lung cancer differential marker havingthe glycan specifically reacting with lectin A can be measured. Examplesof the measurement method include the antibody-overlay lectin arraymethod using a lectin array, LC-MS, immunoassay, enzymatic activityassay, and capillary electrophoresis. Preferably, a qualitative orquantitative approach can be used, which includes: LC-MS; and enzymeimmunoassay, two-antibody sandwich ELISA, gold colloid method,radioimmunoassay, latex agglutination immunoassay, fluorescentimmunoassay, Western blot, immunohistochemical method, surface plasmonresonance spectroscopy (SPR method), and quartz crystal microbalance(QCM) method, using a monoclonal or polyclonal antibody specific for thenovel lung cancer differential marker glycoprotein having the glycanspecifically reacting with lectin A or the fragment thereof.

2-3. Preparation of Specific Polyclonal and/or Monoclonal AntibodiesUsing Lung Cancer Differential Marker Glycoprotein or its Glycopeptide

In the method for detecting lung cancer using the novel lung cancerdifferential marker glycoprotein or its fragment glycopeptide,polyclonal and/or monoclonal antibodies specific for the differentialmarker glycoprotein or its glycopeptide may be used. For example, aneasily obtainable antibody, such as a commercially available antibody,specific for the glycoprotein or the like can be used. If such anantibody is not easily obtainable, it can be prepared, for example, bythe following method:

For example, the anti-lung cancer differential marker glycopeptidepolyclonal antibody can be prepared using a well-known method.Specifically, an adjuvant is added to an obtained antigenic lung cancerdifferential marker glycoprotein or glycopeptide. The antigen used maybe synthesized as a lung cancer differential marker glycopeptidecontaining glycosylation site(s) (asparagine residue(s)). Examples ofthe adjuvant include complete Freund's adjuvant and incomplete Freund'sadjuvant. These adjuvants may be used as a mixture. The antigen may beinoculated, together with the adjuvant, to an antibody-producing animalto thereby boost antibody production. Alternatively, this peptide may becovalently bonded to commercially available keyhole limpet hemocyanin(KLH) and inoculated to an antibody-producing animal. In this procedure,granulocyte-macrophage colony stimulating factor (GM-CSF) may also beadministered to the animal simultaneously therewith to thereby boostantibody production. Examples of the antibody-producing animal that canbe used in antigen inoculation include mammals, for example, mice, rats,horses, monkeys, rabbits, goats, and sheep. Immunization can employ anyof existing methods and is performed mainly by intravenous injection,hypodermic injection, intraperitoneal injection, or the like. Theinterval between immunization doses is not particularly limited and isan interval of several days to several weeks, preferably 4 to 21 days.

2 to 3 days after the final immunization date, whole blood is obtainedfrom the immunized animal. After serum separation, the polyclonalantibody can be prepared.

Alternatively, for example, the anti-lung cancer marker glycopeptidemonoclonal antibody can be prepared by the method of Kohler and Milstein(Nature Vol. 256, pp. 495-497 (1975)). For example, antibody-producingcells obtained from the antigen-immunized animal are fused with myelomacells to prepare hybridomas. From the obtained hybridomas, clonesproducing the anti-lung cancer differential marker glycopeptidemonoclonal antibody can be selected to thereby prepare the monoclonalantibody.

Specifically, 2 to 3 days after the final immunization date in thepreparation of the polyclonal antibody, antibody-producing cells arecollected. Examples of the antibody-producing cells include spleencells, lymph node cells, and peripheral blood cells.

Cell lines that are derived from various animals (e.g., mice, rats, andhumans) and are generally obtainable by those skilled in the art areused as the myeloma cells to be fused with the antibody-producing cells.The cell line used is a drug resistance cell line that cannot survive ina selective medium (e.g., HAT medium) in an unfused state, but cancharacteristically survive therein only in a fused state. In general,8-azaguanine-resistant line is used. This cell line is deficient inhypoxanthine-guanine-phosphoribosyl transferase and cannot grow in ahypoxanthine-aminopterin-thymidine (HAT) medium.

The myeloma cells have already been known in the art, and various celllines can be used preferably, for example, P3(P3x63Ag8.653) (J. Immunol.123, 1548-1550 1979)), P3x63Ag8U.1 (Current Topics in Microbiology andImmunology 81, 1-7 (1978)), NS-1 (Kohler, G. and Milstein, C., Eur. J.Immunol. 6, 511-519 (1976)), MPC-11 (Margulies, D. H. et al., Cell 8,405-415 (1976)), SP2/0 (Shulman, M. et al., Nature 276, 269-270 (1978)),FO (de St. Groth, S. F. et al., J. Immunol. Methods 35, 1-21 (1980)),5194 (Trowbridge, I. S., J. Exp. Med. 148, 313-323 (1978)), and 8210(Galfre, G. et al., Nature 277, 131-133 (1979)).

Next, the myeloma cells and the antibody-producing cells are fused witheach other. This cell fusion is performed by the contact between themyeloma cells and the antibody-producing cells at a mixing ratio of 1:1to 1:10 at 30 to 37° C. for 1 to 15 minutes in the presence of a fusionpromoter in a medium for animal cell culture such as MEM, DMEM, orRPMI-1640 medium. A fusion promoter or a fusion virus, such aspolyethylene glycol or polyvinyl alcohol having an average molecularweight of 1,000 to 6,000 or Sendai virus can be used for promoting thecell fusion. Alternatively, the antibody-producing cells and the myelomacells may be fused with each other using a commercially available cellfusion apparatus based on electrical stimulation (e.g.,electroporation).

After the cell fusion, hybridomas of interest are selected from thefused cells. Examples of the method therefor include a method usingselective growth of the cells in a selective medium. Specifically, thecell suspension is diluted with an appropriate medium and then seededover a microtiter plate. A selective medium (e.g., HAT medium) is addedto each well, and the cells are subsequently cultured with the selectivemedium appropriately replaced by a fresh one. As a result, the cellsthat have grown can be obtained as hybridomas.

The hybridoma screening is performed by, for example, a limitingdilution method or a fluorescence excitation method using a cell sorter.Finally, monoclonal antibody-producing hybridomas are obtained. Examplesof the method for obtaining the monoclonal antibody from the obtainedhybridomas include ordinary cell culture and ascitic fluid formationmethods.

3. Lung Cancer Cell-Identifying Antibody for Histological Staining

The third embodiment of the present invention provides a lung cancercell-identifying antibody for histological staining. The antibody ofthis embodiment is an antibody that can specifically recognize and bindto the lung cancer differential marker according to embodiment 1, i.e.,at least one lung cancer differential marker glycoprotein shown in Table1 or 2 being glycosylated with a glycan at the asparagine residue(s) atthe glycosylation site(s) shown in Table 1 or 2, and/or at least onefragment thereof, the fragment comprising at least one asparagineresidue at the glycosylation site shown in Table 1 or 2 beingglycosylated with a glycan, and thereby specifically identifying a lungcancer cell in histological staining.

The epitope of the lung cancer differential marker according toembodiment 1 recognized by the antibody of this embodiment is notparticularly limited and is preferably a core protein or a fragment ofthe core protein, more preferably a moiety encompassing both of a glycanand its neighboring peptide sequence. In this case, the length of thepeptide sequence is 5 to 15 amino acids, 5 to 10 amino acids, or 5 to 8amino acids. In addition to this antibody probe, a phage antibody probeprepared using a phage can also be used as a glycan probe foridentification of a lung cancer cell.

The antibody of this embodiment is capable of determining thehistological type of a lung cancer cell. Examples of such an antibodyinclude an antibody (R&D Systems, Inc., Anti-human Neuronal PentraxinReceptor Antibody) against neuronal pentraxin receptor (NPR) representedby Protein #21 in Table 1. This anti-neuronal pentraxin receptorantibody is capable of specifically identifying a small cellcancer-derived cell, as shown in FIG. 7, and as such, can serve as anantibody for identification of a small cell cancer cell. Thus, theproteins collected and identified with cancer-specific glycan probelectins are usually expressed at an exceedingly low level at othertissues or normal sites and thus include tissue markers that canfunction by means of only the antibody or the phage antibody without theneed of differentiating between small cell cancer-derived cells and lungadenocarcinoma-derived cells using lectins or the like.

EXAMPLES Example 1 Selection of Glycopeptide Marker by GlycoproteomicsIGOT-LC/MS Method 1. Method for Preparing Culture Supernatant of HumanLung Cancer Cell

Three lung adenocarcinoma cell lines (H358, H1975, and LX-1) and threesmall cell lung cancer cell lines (H2171, H524, and H526) wereseparately cultured for 3 days using a high-glucose medium containing90% DMEM and 10% FBS (PS+) in a dish having a diameter of 14 cm toachieve 80 to 90% confluence. The FBS-containing medium was discarded byaspiration, and the cells of each line were washed with 10 mL/dish of aserum-free medium (100% DMEM-high glucose, no additive). After additionof a serum-free medium at a concentration of 30 mL/dish, the cells werecultured for 48 hours. The cells thus cultured were centrifuged at 1000rpm for 30 minutes to recover a supernatant. The supernatant (culturesupernatant) was further recovered by centrifugation at 3000 rpm for 30minutes and cryopreserved at −80° C. The culture supernatant stored atthis temperature was thawed before use, filtered through a 0.45-μmfilter, and then used in Examples below. NaN₃ was added to each culturesupernatant to adjust the final concentration to 0.1%.

2. Method for Identifying Glycoprotein at Large Scale

(1) Preparation of Peptide Sample

Trichloroacetic acid (TCA, 100% saturated aqueous solution) was added ata final concentration of 10% to each culture supernatant thus prepared.The mixture was cooled on ice for 10 to 60 minutes to precipitateproteins. The precipitates were recovered by centrifugation at a highspeed at 4° C. The precipitates were suspended in ice-cold acetone andwashed twice to remove TCA. A lysis buffer solution (containing 0.5 Mtris-HCl buffer solution, pH 8-8.5, 7 M guanidine hydrochloride, and 10mM EDTA) was added to the precipitates to adjust the proteinconcentration to 5 to 10 mg/mL, while the proteins were lysed therein.In another method, each culture supernatant was applied to anultrafiltration membrane having a molecular weight cutoff of 10,000 at4° C. to concentrate proteins, to which a lysis buffer solution was thenadded. The protein solution was filtered again to prepare a sampleprotein solution.

Extracts were recovered from each supernatant by centrifugation at ahigh speed. Nitrogen gas was passed through or sprayed to the extractsto remove dissolved oxygen. Then, dithiothreitol (DTT) in the form of apowder or dissolved in a small amount of a lysis buffer solution wasadded thereto in an amount equal to the protein weight. The mixture wasreacted at room temperature for 1 to 2 hours with nitrogen gas passedtherethrough or in a nitrogen gas atmosphere to reduce the disulfidebond. Subsequently, iodoacetamide for S-alkylation was added thereto inan amount of 2.5 times the protein weight. The mixture was reacted atroom temperature for 1 to 2 hours in the dark. The reaction solution wasdialyzed at 4° C. (cooling room) against a 50- to 100-fold amount of abuffer solution (in general, 10 mM ammonium bicarbonate buffer solution,pH 8.6) as an external solution. The external solution was replaced by afresh one three to five times at appropriate time intervals to removethe denaturant (guanidine hydrochloride) or an excess of the reagents.Although proteins were partially precipitated, this suspension wassubjected directly to protein quantification. Trypsin (sequencing gradeor higher) with a weight of 1/100 to 1/50 of the protein amount wasadded thereto to digest the proteins overnight (approximately 16 hours)at 37° C. The progression of the digestion was confirmed by SDS-gelelectrophoresis. When sufficient digestion was confirmed, the reactionwas terminated by the addition of phenylmethanesulfonyl fluoride (PMSF)at a final concentration of 5 mM.

(2) Collection and Purification of Candidate Glycopeptide

The sample peptides prepared in the preceding step were applied to probelectin (AAL lectin or ConA lectin)-immobilized columns. After washing,candidate glycopeptides were eluted by a method appropriate for thespecificity of each lectin, i.e., using a buffer solution containing 5mM fucose as to the AAL lectin and using a buffer solution containing0.2 M methylmannoside as to the ConA lectin. To the obtained candidateglycopeptide solution, an equal volume of ethanol and a 4-fold volume of1-butanol were added, and the mixture was applied to a Sepharose columnequilibrated in advance with water:ethanol:1-butanol (1:1:4 (v/v)). Thecolumn was washed with this equilibrating solvent, and candidateglycopeptides were then eluted with 50% ethanol (v/v). Each candidateglycopeptide fraction was transferred in small portions to a microtubecontaining 2 μl, of glycerol and concentrated by centrifugation underreduced pressure (i.e., water was removed by centrifugation underreduced pressure). This procedure was repeated to concentrate allcandidate glycopeptide fractions.

(3) Glycan Cleavage and Isotope-Coded Glycosylation Site-SpecificTagging (IGOT) Method

A necessary amount of a buffer solution was added to the purifiedcandidate glycopeptides (glycerol solution), and the mixture wasconcentrated again by centrifugation under reduced pressure. Then,stable oxygen isotope-18 (¹⁸O)-labeled water (H₂ ¹⁸O) was added theretoto dissolve the concentrate (glycerol concentration: 10% or lower).Peptide-N-glycanase (glycopeptidase F, PNGase) prepared with labeledwater was added thereto and reacted overnight at 37° C. This reactioncauses the conversion of the glycosylated asparagine to aspartic acid,during which the oxygen isotope (¹⁸O) in the water is incorporated intothe candidate glycopeptide to label the candidate glycopeptide.

(4) LC/MS Shotgun Analysis of Labeled Peptide

The IGOT reaction solution was diluted with 0.1% formic acid andsubjected to LC/MS shotgun analysis. In this analysis, a nano-LC systembased on a direct nano-flow pump was used for high-separation,high-reproducibility, and high-sensitivity detection. The injectedcandidate glycopeptides were temporarily collected onto a trap column(reverse-phase C18 silica gel carrier) intended for desalting. Afterwashing, the glycopeptides were separated by the concentration gradientof acetonitrile using frit-less spray tip nano-columns (inside diameter:150 μm×50 mL) packed with the same resins. The eluate was ionized via anelectrospray interface and directly introduced into a mass spectrometer.The glycopeptides were analyzed by collision-induced dissociation(CID)-tandem mass spectrometry in a data-dependent mode in which twoions at the maximum to be analyzed were selected.

(5) Search for Candidate Glycopeptide by MS/MS Ion Search Method

Thousands of MS/MS spectra thus obtained were individually smoothed andconverted to centroid spectra to prepare peak lists. On the basis of thepeak lists, each candidate glycopeptide was identified by the MS/MS ionsearch method using a protein amino acid sequence database. The searchengine used was Mascot (Matrix Science Ltd.). The following parameterswere used for search conditions: a fragmentation method used: trypsindigestion, the maximum number of missed cleavage: 2, fixed modification:carbamidomethylation of cysteine, variable modifications: deamination ofan N-terminal amino group (N-terminal glutamine), oxidation ofmethionine, ¹⁸O-incorporating deamidation of asparagine (glycosylationsite), error tolerance of MS spectrum: 500 ppm, and error tolerance ofMS/MS spectrum: 0.5 Da.

(6) Identification of Candidate Glycopeptide

The database was searched under the conditions described above. Theobtained identification results were validated according to criteriashown below. The obtained candidate glycopeptides were regarded as lungcancer differential marker glycopeptides (lung cancer differentialmarker glycoprotein fragments).

(i) The probability score (coincidence probability: Expectation value)of identification is 0.05 or less.

(ii) The number of fragment ions contributing to identification is 4 ormore.

(iii) Error (ppm) is not significantly deviated from systematic error(mass error being 0.5 Da or less).

(iv) Each identified peptide has consensus sequence(s) with the numberof Asn modifications (conversion to Asp and incorporation of ¹⁸O) equalto or fewer than the number of the consensus sequence(s).

(7) Identification of Lung Cancer Differential Marker Glycoprotein

Glycoproteins containing the sequences of the selected lung cancerdifferential marker glycopeptides can be identified using thesesequences. More specifically, the whole amino acid sequences ofcorresponding lung cancer differential marker glycoproteins wereidentified from the amino acid sequence database NCBI-Refseq on thebasis of the “peptide sequences” of lung cancer differential markerglycopeptides represented by SEQ ID NOs: 1 to 223 in Tables 1 and 2.

(Results)

The lung cancer differential marker glycoproteins obtained by the stepsdescribed above are shown in Table 2.

3. Validation of Culture Supernatant-Derived Protein by Immunoblot

Each of the culture supernatants of lung cancer cell lines (three lungadenocarcinoma cell lines: H358, H1975, and LX-1, and three small celllung cancer cell lines: H2171, H524, and H526) separately cultured for24 hours in a serum-free medium was concentrated 10-fold through AmiconUltra-154 Centrifugal Filter Units (cutoff: 10 kDa, Millipore Corp.).Each sample (10 μL) was developed by SDS-PAGE on a 10%, 12.5%, or 17%acrylamide gel of XV PANTERA SYSYTEM (Maruko Shokai Co., Ltd.) and thentransferred to a PVDF membrane (GE Healthcare Japan Corp.) at 200 mA for40 min. The blocking agent used was PBSTx (Dulbecco's PBS supplementedwith 1% Triton X-100) containing 5% skimmed milk or 5% BSA dissolvedtherein. The membrane was blocked at room temperature for 1 hour. After10-minute washing three times with PBSTx, the membrane was reacted for 1hour with primary antibodies (Table 4) biotinylated in advance withBiotin Labeling Kit-NH₂ (Dojindo Laboratories). After 10-minute washingthree times with PBSTx again, the membrane was reacted for 1 hour withsecondary antibody-HRP-conjugated streptavidin (1:3000 dilution, GEHealthcare Japan Corp.). After 10-minute washing three times with PBSTx,the enzymatic reaction of HRP was caused using Western LightningChemiluminescence Reagent Plus (PerkinElmer, Inc.). The obtained signalswere developed onto Amersham Hyperfilm ECL (GE Healthcare Japan Corp.).The amounts of proteins present in the culture supernatants derived fromthe lung adenocarcinoma and small cell lung cancer cell lines wereanalyzed by comparison.

TABLE 4 Antibody # Antibody name [Manufacturer: Catalog No./Distributer]01 neural cell adhesion molecule 1 (NCAM-1/CD56) [LSP:MAB24081/Funakoshi Corp.] 02 neuronal pentraxin II (NPTX2) [LSP:LS-C53292-50/Funakoshi Corp.] 03 Thy-1 cell surface antigen [ABV:H00007070-M01/Cosmo Bio Co., Ltd.] 04 insulin-like growth factor 2receptor (IGF-IIR) [RSD: AF2447/Cosmo Bio Co., Ltd.] 05 insulin-likegrowth factor 2 receptor (IGF-IIR) [RSD: MAB2447/Funakoshi Corp.] 06acid alpha-glucosidase preproprotein (GAA) [LSP: LS-C80648-50/Cosmo BioCo., Ltd.] 07 sparc/osteonectin, cwcv and kazal-like domainsproteoglycan 2 (testican2/SPOCK2) [RSD: MAB2328/Funakoshi Corp.] 08sparc/osteonectin, cwcv and kazal-like domains proteoglycan 2(testican2/SPOCK2) [ABV: H00009806-B01P/Funakoshi Corp.] 09 celladhesion molecule 4 (CADM4) [LSP: LS-C36881/Cosmo Bio Co., Ltd.] 10secretogranin III (SgIII) [SCB: SC-1492/Cosmo Bio Co., Ltd.] 11melanoma-associated antigen p97 (MFI2) [ABV: H00004241-B01P/Cosmo BioCo., Ltd.] 12 cathepsin L2 preproprotein (CTSL2) [ABV: PAB8639/Cosmo BioCo., Ltd.] 13 cathepsin L2 preproprotein (CTSL2) [DFK: F-106/Cosmo BioCo., Ltd.] 14 lysosomal acid phosphatase 2 precursor (ACP2) [ABV:PAB7218/Cosmo Bio Co., Ltd.] 15 lysosomal acid phosphatase 2 precursor(ACP2) [ABV: H00000053-M01/Cosmo Bio Co., Ltd.] 16 cathepsin L1 (CTSL1)[ABV: PAB8638/Cosmo Bio Co., Ltd.] 17 cathepsin L1 (CTSL1) [ABV:MAB1432/Cosmo Bio Co., Ltd.] 18 deoxyribonuclease II, lysosomalprecursor (DNASE2) [ABV: H00001777-B01P/Cosmo Bio Co., Ltd.] 19 integralmembrane protein 1 (ITM1) [SCB: SC-100796/Funakoshi Corp.] 20 integralmembrane protein 1 (ITM1) [ABV: H00003703-M02/Cosmo Bio Co., Ltd.] 21neuronal pentraxin receptor (NPTXR/NPR) [RSD: AF4414/Funakoshi Corp.] 22neuronal pentraxin receptor (NPTXR/NPR) [LSP: LS-C73727-100/FunakoshiCorp.] 23 neuronal pentraxin receptor (NPTXR/NPR) [SCB: SC-12483/CosmoBio Co., Ltd.] 24 sushi domain containing 2 (SUSD2) [ABV:H00056241-B01/Cosmo Bio Co., Ltd.] 25 sel-1 suppressor of lin-12 like(SEL1L) [LSP: LS-B2253-50/Funakoshi Corp.] 26 sel-1 suppressor of lin-12like (SEL1L) [LSP: LS-C55443-100/Funakoshi Corp.] 27 sel-1 suppressor oflin-12 like (SEL1L) [ABV: PAB7473/Cosmo Bio Co., Ltd.] 28 source ofimmunodominant MHC-associated peptides (STT3B) [PG: 15323-1-AP/Cosmo BioCo., Ltd.] 29 IGFBP-L1 [RSD: AF3877/Cosmo Bio Co., Ltd.] 30 neural celladhesion molecule 1 (NCAM-1/CD56) [SCB: sc-71647/Cosmo Bio Co., Ltd.] 31Fibronectin (H-300) [SCB: sc-9068/Cosmo Bio Co., Ltd.] 32Galectin-3BP/MAC-2BP (MAC2BP) [RSD: AF2226/Cosmo Bio Co., Ltd.] 33Cathepsin D [RSD: AF1014/Cosmo Bio Co., Ltd.] 34 Fibronectin (C-20)[SCB: sc-6952/Cosmo Bio Co., Ltd.] 35 neogenin homolog 1 (NGN) [SCB:SC-15337]/Cosmo Bio Co., Ltd.] 36 laminin alpha 5 [SCB: SC-20145/CosmoBio Co., Ltd.] 37 laminin, beta 1 precursor [SCB: sc-17763/Cosmo BioCo., Ltd.] 38 phospholipid transfer protein isoform b precursor (PLTP)[RSD: AF5109/Cosmo Bio Co., Ltd.] 39 melanoma cell adhesion molecule(MCAM) [Millipore: MAB16985/] 40 L1 cell adhesion molecule isoform 2precursor (L1CAM) [LSP: LS-C49042/Cosmo Bio Co., Ltd.] 41 biotinidaseprecursor (BTD) [ABV: H00000686-M01/Funakoshi Corp.] 42 ribonuclease T2precursor (RNASET2) [ABV: H00008635-B01/Cosmo Bio Co., Ltd.] 43tubulointerstitial nephritis antigen-like 1 (TINAGL1) [ABV:H00064129-B02P/Cosmo Bio Co., Ltd.] 44 v-kit Hardy-Zuckerman 4 felinesarcoma viral oncogene homolog isoform 2 precursor (KIT) [LSP:LS-C40873-100/Cosmo Bio Co., Ltd.] 45 secretogranin III (SgIII) [SCB:SC-50289] 46 secretogranin III (SgIII) [SIGMA: HPA-006880] 47 IGFBP-L1[RSD: BAF3877/Cosmo Bio Co., Ltd.]

(Results)

The lung cancer differential marker glycoproteins obtained by the stepsdescribed above are shown in Table 1 and FIGS. 1 and 2.

4. Batch Fractionation of Culture Supernatant Using AAL Lectin

The culture supernatants were fractionated using AAL lectin.Specifically, an AAL-conjugated resin was washed five times with a3-fold amount of PBS and then prepared into a 50% slurry solution. To 30μL of the prepared AAL-conjugated resin, 30 μL of each culturesupernatant was added, and the resin was shaken at 4° C. for 5 hours.After centrifugation (2,000 rpm, 2 min.), the supernatant was removed,and 50 μL of a wash buffer (0.1% SDS in PBST, and 0.1% Triton X-100) wasthen added to the resulting resin. After centrifugation (2,000 rpm, 2min.) two times, 500 μL of a wash buffer was added thereto. Thesupernatant was removed by centrifugation (2,000 rpm, 2 min.), and theresin was washed. For elution from the washed resin, 15 μL of 0.2 Mfucose in PBS containing 0.02% SDS was added thereto, and the resin wasshaken at 4° C. for 5 hours. After centrifugation (2,000 rpm, 2 min.),the supernatant was recovered. 15 μL of an elution buffer was furtheradded thereto, and the resin was centrifuged (2,000 rpm, 2 min.) toelute AAL-bound fractions. 10 μL each of the eluted fractions wasdeveloped by SDS-PAGE, and proteins were detected by Western blot.Similar procedures were performed using ConA lectin except that 0.5 Mmethylmannoside was used in elution.

(Results)

The lung cancer differential marker glycoproteins obtained by the stepsdescribed above are shown in FIG. 3.

5. Fractionation by Immunoprecipitation

Antibodies against all the candidate glycoproteins shown in Table 1 werebiotinylated, if unbiotinylated, using Biotin Labeling Kit-NH₂ (DojindoLaboratories) according to the manual. 1 μg of the biotinylatedantibodies was added to 100 μL of each culture supernatant and shaken at1,400 rpm at 20° C. for 2 hours. For washing of magnetic beads(Invitrogen Corp.), 100 μL of TBSTx (50 mM Tris-HCl (pH 8.0), 150 mMNaCl, and 1% Triton X-100) was added to 20 μL of the magnetic beads andstirred, followed by supernatant removal three times by centrifugation(10,000 rpm, 3 sec.). The reacted culture supernatant and antibodieswere transferred to the magnetic beads and shaken at 1,400 rpm at 20° C.for 1 hour. The antibodies bound with the magnetic beads were recoveredusing a magnetic stand. The recovered magnetic beads were washed threetimes with 1 mL of TBSTx. After addition of 20 ul of an elution buffer(0.2% SDS in TBS), the mixture was stirred and then heated two times at98° C. or 60° C. for 5 minutes for elution. In order to remove theantibodies on the eluted sample, 40 μL of magnetic beads washed in thesame way as above was added to the eluted sample and shaken at 1,400 rpmat 20° C. for 2 hours. The antibody-bound magnetic beads were removedusing a magnetic stand, and the remaining portion was used as a sample.The prepared sample was developed by SDS-PAGE and then subjected tolectin blot.

(Results)

The lung cancer differential marker glycoproteins obtained by the stepsdescribed above are shown in FIG. 4.

6. Evaluation by Lectin Blot

10 μL of the immunoprecipitated sample was developed on a 10% gel bySDS-PAGE. After SDS-PAGE, the proteins were transferred to a PVDFmembrane (GE Healthcare Japan Corp.). The PVDF membrane was blocked with5% BSA at room temperature for 1 hour and then reacted with alreadybiotinylated AAL lectin (Seikagaku Corp.) at room temperature for 1hour. Then, the membrane was reacted with secondaryantibody-HRP-conjugated streptavidin at room temperature for 1 hour. Theproteins were detected using Western Lightning. PBS-T was used in thedilution of lectin.

(Results)

The lung cancer differential marker glycoproteins obtained by the stepsdescribed above are shown in FIG. 4.

7. Column Fractionation of Serum Using AAL Lectin

Serum was fractionated using AAL lectin. Specifically, a commerciallyavailable column was packed with 1 mL of an AAL-conjugated resin andwashed with TBS in an amount of 10 times the amount of the resin, with0.5 M NaCl in an amount of twice the amount of the resin, and with TBSin an amount of 10 times the amount of the resin to prepare an AALcolumn. Serum was added to the AAL column and then reacted for 1 hour inthe resin. Then, the column was washed with TBS in an amount of 5 timesthe amount of the resin. For elution, 1 mL of 20 mM fucose was addedthereto and reacted for 1 hour in the resin. Then, 4 mL of 20 mM fucosewas further added thereto (a total of 5 mL) to elute the proteins.

(Results)

The lung cancer differential marker glycoproteins obtained by the stepsdescribed above are shown in FIG. 5.

8. Fractionation of Serum by Serial Chromatography

Serum was fractionated at a low temperature by serial chromatographyusing LCA agarose (J-Oil Mills, Inc.) and AAL agarose (J-Oil Mills,Inc.). Specifically, 100 μL of serum was diluted 4-fold with PBS. Theserum sample was first applied to a column packed with 5 mL of LCAagarose (0.7×13 cm). Next, the obtained unbound serum fractions wereapplied to a column packed with 2.5 mL of AAL agarose (0.7×5.5 cm).After sufficient washing with PBS, the proteins were eluted with PBScontaining 0.2 M fucose. The eluted fractions were concentrated throughUltrafree Centrifugal Filter Device (cutoff: 30 kDa, Millipore Corp.).The candidate molecules in the obtained samples were analyzed bycomparison by immunoblot in the same way as above.

(Results)

The lung cancer differential marker glycoproteins obtained by the stepsdescribed above are shown in FIG. 6.

9. Multisample Fractionation of Serum by Serial Chromatography

The multisample comparative analysis of sera requires fractionating asmall amount of a serum sample in a short time. For this purpose,high-throughput serial chromatography was established. Sera werefractionated by serial chromatography using LCA agarose (J-Oil Mills,Inc.) and AAL agarose (Vector Laboratories, Inc.). 50 μL of a serumsample was applied to a tip column packed with 300 μL of LCA agarose(0.37×2.3 cm). Next, the obtained unbound serum fractions were appliedto an open column packed with 250 μL of AAL agarose (0.8×0.5 cm). Aftersufficient washing with PBS, the proteins were eluted with PBScontaining 0.02 M fucose. The eluted fractions were concentrated throughUltrafree Centrifugal Filter Device (cutoff: 30 kDa, Millipore Corp.).The candidate molecules in the obtained samples (corresponding to 10 μLof sera) were analyzed by comparison by immunoblot in the same way asabove.

(Results)

The lung cancer differential marker glycoproteins obtained by the stepsdescribed above are shown in FIG. 8.

10. Antibody-Overlay Lectin Array

An appropriate amount of the glycoprotein solution obtained in theparagraph “5. Fractionation by immunoprecipitation” was adjusted to 60μL with PBSTx (phosphate-buffered saline containing 1% Triton X-100) asa lectin array reaction buffer. This solution was added to each reactionvessel of a lectin array composed of 8 reaction vessels per a singleglass plate, and reacted at 20° C. for 10 hours or longer. This lectinarray substrate composed of 8 reaction vessels was prepared according tothe approach of Uchiyama et al. (Proteomics 8, 3042-3050 (2008)). Inthis way, the binding reaction between the glycans on the glycoproteinsand 43 types of lectins immobilized on the array substrate reaches anequilibrium state. In order to prevent noise from being generated by thebinding of the glycans of antibodies for detection to unreacted lectinson the substrate, 2 μL of a human serum-derived IgG solution(manufactured by Sigma-Aldrich Corp.) was then added and reacted for 30minutes. Each reaction vessel was washed three times with 60 μL ofPBSTx. Then, 2 μL of a human serum-derived IgG solution was addedthereto again and slightly stirred. Subsequently, biotinylatedantibodies for detection against the glycoproteins were added in anamount corresponding to 100 ng and reacted at 20° C. for 1 hour. Afterthis antigen-antibody reaction, each reaction vessel was washed threetimes with 60 μl, of PBSTx. Then, the array was scanned with an arrayscanner GlycoStation manufactured by Moritex Corporation to comparefluorescence intensities on the lectin spots reacted in a lung cancertissue-specific manner.

(Results)

The lung cancer differential marker glycoproteins obtained by the stepsdescribed above are shown in FIGS. 9 and 10.

The results of these experiments revealed that some lectins, such as PNAand PWM, other than AAL or ConA and lectins having specificity relatedto these ligands are effective for the differential diagnosis of lungcancer. This means that a plurality of lectin signals derived fromglycoproteins may be combined in various ways to thereby more accuratelydetermine the histological type of lung cancer.

Example 2 Histological Staining of Lung Cancer Cell Using Lung CancerDifferential Marker

Histological staining with antibodies was tested using lung cancerdifferential markers.

First, paraffin that covered a formalin-fixed lung cancer tissue section(5 μm thick) was removed according to a standard method. Thedeparaffinized tissue section was washed with PBS, dried in air, andthen dipped in a 10 mM citrate buffer. The intermolecular(intramolecular) bridges derived from formalin fixation were dissociatedby autoclaving at 121° C. for 15 minutes. The section thus treated wasleft standing at room temperature for a while and then dipped threerepetitive times in PBS for 5 minutes to wash the surface of the tissue.Subsequently, the section was treated with 0.3% H₂O₂-MeOH at roomtemperature for 10 minutes for the blocking reaction of endogenousperoxidase. After washing with PBS (5 min.×3), a primary antibodysolution (R&D Systems, Inc.; anti-NPR antibody suspended at aconcentration of 3 μg/mL in PBS) was added onto the tissue section tocause binding reaction at 20° C. for 2 hours in a humidifying box. Afterwashing with PBS (5 min.×3), chromogenic reaction was initiated using ananti-sheep FITC conjugate (Santa Cruz Biotechnology, Inc.) as asecondary antibody and an anti-FITC HRP conjugate (Takara Bio Inc.) asan enzymatically labeled antibody and terminated by dipping in Milli-Qwater for 5 minutes three times. Finally, the nucleic acids were stainedwith hematoxylin at room temperature for 1 minute, followed by washingin running water.

(Results)

The lung cancer differential marker glycoproteins obtained by the stepsdescribed above are shown in FIG. 7. Staining specific only for smallcell cancer was confirmed.

All publications, patents, and patent applications cited herein areincorporated herein by reference in their entirety.

1. A lung cancer differential marker glycoprotein listed in at least oneof Table 1 or Table 2, being glycosylated with a glycan at an asparagineresidue at a glycosylation site shown in Table 1 or Table 2, as follows:TABLE 1  Small cell cancer Adenocarcinoma Glycosylation SEQ Protein #Protein name gi(ID) ML ConA ML ConA site Peptide sequence NO. 1acid alpha-glucosidase gi|119393891, ◯ X X X 390 QWENMTR 1 gi|119393893470 GVFITNETGQPLIGK 2 882 GAYTQVIFLARNNTIVNELVR 3 2 biotinidasegi|4557373 X X ◯ X 119 DVQIIVFPEDGIHGFNFTR 4 150 FNDTEVLQR 5 349SHLIIAQVAKNPVGLIGAENATGETDPSHSK 6 349 NPVGLIGAENATGETDPSHSKFLK 7 3cathepsin D gi|4503143 X X ◯ X 263 YYKGSLSYLNVTR 8 4 cathepsin L1gi|22202619, ◯ X X X 221 YNPKYSVANDTGFVDIPKQEK 9 gi|4503155 221YSVANDTGFVDIPK 10 5 cathepsin L2 gi|23110960 ◯ X X X 221YRPENSVANDTGFTWAPGKEK 11 292 NLDHGVLVVGYGFEGANSNNSK 12 6cell adhesion molecule 4 gi|21686977 ◯ X X X 67 QTLFFNGTR 13 7deoxyribonuclease II, gi|4503349 ◯ X X X 86 SNTSQLAFLLYNDQPPQPSK 14lysosomal 8 fibronectin 1 gi|47132557 (isoform 1), X X ◯ X 430GGNSNGALCHFPFLYNNHNYTDCTSEGR 15 gi|47132551 (isoform 2), 528DQCIVDDITYNVNDTFHK 16 gi|16933542 (isoform 3), 542 RHEEGHMLNCTCFGQGR 17gi|47132555 (isoform 4), 542 HEEGHMLNCTCFGQGR 18gi|47132553 (isoform 5), 1007 ESKPLTAQQTTKLDAPTNLQFVNETDSTVLVR 19gi|47132549 (isoform 6), 1007 LDAPTNLQFVNETDSTVLVR 20gi|47132547 (isoform 7) 1291 WTPLNSSTIIGYR 21 9 galectin-3-bindinggi|122937327 ◯ X X X 44 ADVGGEAAGTSINHSQAVLQR 22 protein-like 61QGNASDWLR 23 307 FFDVNGSAFLPR 24 10 insulin-like growth factor 2gi|119964726 ◯ X X X 112 SLLEFNTTVSCDQQGTNHR 25 receptor 435MSVINFECNKTAGNDGK 26 582 TNITLVCKPGDLESAPVLR 27 2085GYPCGGNKTASSVIELTCTK 28 11 insulin-like growth factorgi|62243248 (isoform a), X X ◯ X 116 GLCVNASAVSR 29 binding protein 3gi|62243068 (isoform b) 205 YKVDYESQSTDTQNFSSESKR 30 12insulin-like growth factor gi|56090548 X ◯ X X 166DGPCEFAPVWVPPRSVHNVTGAQVGLSCEVR 31 binding protein-like 1 166SVHNVTGAQVGLSCEVR 32 13 integral membrane protein 1 gi|22749415 X ◯ X X548 TILVDNNTWNNTHISR 33 14 L1 cell adhesion moleculegi|4557707 (isoform 1), X X ◯ ◯ 433 ILTADNQTYMAVQGSTAYLLCK 34  gi|13435353 (isoform 2) 671 WYSLGKVPGNQTSTTLK 35 777VQWRPQGTRGPWQEQIVSDPFLVVSNTSTFVPYEIK 36 979 THNLTDLSPHLR 37 15lysosomal acid phosphatase 2 gi|4557010 ◯ X X X 133FNPNISWQPIPVHTVPITEDR 38 167 YEQLQNETRQTPEYQNESSR 39 177 QTPEYQNESSR 4016 melanoma cell adhesion gi|1274107 ◯ X X X 56 CGLSQSQGNLSHVDWFSVHK 41molecule 418 CVASVPSIPGLNR 42 17 melanoma-associated antigengi|134244281 (isoform 1), ◯ ◯ X X 38 WCATSDPEQHKCGNMSEAFR 43 p97gi|16163666 (isoform 2) 515 DCDVLTAVSEFFNASCVPVNNPK 44 18neogenin homolog 1 gi|4505375 X ◯ X X 73 GSSVILNCSAYSEPSPK 45 210VIKLPSGMLVISNATEGDGGLYR 46 470 TPASDPHGDNLTYSVFYTK 47 19neural cell adhesion gi|94420689 (isoform 1), ◯ X X X 347 TSTRNISSEEK 48molecule 1 gi|115529482 (isoform 2), 449 DGQLLPSSNYSNIK 49gi|115529478 (isoform 3) 478 IYNTPSASYLEVTPDSENDFGNYNCTAVNR 50 20neuronal pentraxin II gi|28195384 ◯ X X X 148 ANVSNAGLPGDFR 51 189VAELEDEKSLLHNETSAHR 52 21 neuronal pentraxin receptor gi|17402888 X ◯ XX 42 ALPGGADNASVASGAAASPGPQR 53 22 ribonuclease T2 gi|5231228 X X ◯ X106 AYWPDVIHSFPNR 54 212 QDQQLQNCTEPGEQPSPK 55 23 secretogranin IIIgi|19557645 ◯ ◯ X X 68 KTYPPENKPGQSNYSFVDNLNLLK 56 346 NKLEKNATDNISK 5724 sel-1 suppressor of gi|19923669 X ◯ X X 608 EASIVGENETYPR 58lin-12-like 25 sparc/osteonectin, cwcv and gi|7662036 ◯ ◯ X X 225LRDWFQLLHENSKQNGSASSVAGPASGLDK 59 kazal-like domains  225QNGSASSVAGPASGLDK 60 proteoglycan (testican) 2 26Thy-1 cell surface antigen gi|19923362 ◯ ◯ X X 42 LDCRHENTSSSP1QYEFSLTR61 27 tubulointerstitial nephritis gi|11548918 X X ◯ X 78GRADDCALPYLGAICYCDLFCNR 62 antigen-like 1 161AINQGNYGWQAGNHSAFWGMTLDEGIR 63 28 v-kit Hardy-Zuckerman 4gi|4557695 (isoform 1), ◯ ◯ X X 130 SLYGKEDNDTLVR 64feline sarcoma viral gi|148005039 (isoform 2) 367 TFTDKWEDYPKSENESNIR 65oncogene homolog 463 CSASVLPVDVQTLNSSGPPFGK 66 486 LVVQSSIDSSAFKHNGTVECK67 29 laminin alpha 5 gi|21264602 X X ◯ ◯ 95 LVGGPVAGGDPNQTIR 68 921LNLTSPDLFWLVFR 69 1330 VWQGHANASFCPHGYGCR 70 1529TIPPDCLLCQPQTFGCHPLVGCEECNCSGPGIQELT 71 DPTCDTDSGQCK 2019CEICAPGFYGNALLPGNCTR 72 2196 GINASSMAWAR 73 2209 LHRLNASIADLQSQLR 742303 TLSELMSQTGHLGLANASAPSGEQLLR 75 2423 DNATLQATLHAAR 76 2501LVEAAEAHAQQLGQLALNLSSIILDVNQDRLTQR 77 2568 QGLVDRAQQLLANSTALEEAMLQEQQR78 2707 GVHNASLALSASIGR 79 3107 LNTTGVSAGCTADLLVGR 80 3287VFDLQQNLGSVNVSTGCAPALQAQTPGLGPR 81 30 laminin, beta 1 gi|4504951 X X ◯ ◯1041 KCVCNYLGTVQEHCNGSDCQCDK 82 1279 LSDTTSQSNSTAK 83 1487QSAEDILLKTNATK 84 1643 AIKQADEDIQGTQNLLTSIESETAASEETLFNASQR 85 31phospholipid transfer gi|5453914 (isoform a), X X ◯ ◯ 64 GKEGHFYYNISEVK86 protein gi|33356541 (isoform b) 143 MKVSNVSCQASVSR 87 143VSNVSCQASVSR 88 245 GAFFPLTERNWSLPNR 89 398 FRIYSNHSALESLALIPLQAPLK 90193 GAFFPLTERNWSLPNR 91

TABLE 2  Small cell cancer Adenocarcinoma Glycosylation SEQ Protein #Protein name gi(ID) AAL ConA ML ConA site Peptide sequence NO. 1activated leukocyte cell gi|68163411 X ◯ X X 167 KLGDCISEDSYPDGNITWYR 92adhesion molecule 265 NAIKEGDNITLK 93 265 EGDNITLK 94 361 NATVVWMKDNIR95 480 YYSKIIISPEENVTLTCTAENQLER 96 2 alpha 2 type V collagengi|89363017 X X X ◯ 1400 EASQNITYICK 97 preproprotein 3amiloride binding gi|73486661 X X ◯ ◯ 538 LENITNPWSPR 98 protein 1 4aspartate beta-hydroxylase gi|14589866 (isoform a) X X X ◯ 452LVQLFPNDTSLKNDLGVGYLLIGDNDNAKK 99 5 aspartate beta-hydroxylasegi|14589864 (isoform b) ◯ ◯ X X 64 DFRYNLSEVLQGK 100 6beta-1,3-galactosyl-O- gi|148277029, X X ◯ X 58 HLELAGENPSSDINCTK 101glycosyl-glycoprotein gi|148277031, 95 WTPDDYINMTSDCSSFIK 102beta-1,6-N-acetyl- gi|148277033, glucosaminyltransferase gi|148277035,gi|21614523 7 bone morphogenetic gi|4502421 X X ◯ ◯ 142AATSRPERVWPDGVIPFVIGGNFTGSQR 103 protein 1 363 ISVTPGEKIILNFTSLDLYR 104599 LNGSITSPGWPK 105 8 calsyntenin 2 gi|11545861 ◯ X X X 98IHGQELPFEAVVLNKTSGEGR 106 374 NLTDQFTITMWMK 107 716 QECLELNHSELHQR 108729 HLDATNSTAGYSIYGVGSMSR 109 9 carboxypeptidase D gi|22202611 X ◯ X X172 LLNTTDVYLLPSLNPDGFERAR 110 522 RFANEYPNITR 111 978HIWSLEISNKPNVSEPEEPKIR 112 1070 GKDLDTDFTNNASQPETK 113 10 CD47 antigengi|4502673 (isoform 1), X ◯ X X 73 GRDIYTFDGALNK 114gi|38683836 (isoform 2), 73 DIYTFDGALNKSTVPTDFSSAK 115gi|68223315 (isoform 3) 111 MDKSDAVSHTGNYTCEVTELTR 116 11 CD63 antigengi|4502679 (isoform A), X ◯ X X 130 QQMENYPKNNHTASILDR 117gi|91199546 (isoform B) 130 NNHTASILDR 118 172 NRVPDSCCINVTVGCGINFNEK119 12 CD97 antigen gi|17978491 (isoform 1), X X ◯ X 108TFKNESENTCQDVDECQQNPR 120 gi|17978489 (isoform 2), 453 RLSAVNSIFLSHNNTK121 gi|68508955 (isoform 3) 360 RLSAVNSIFLSHNNTK 122 108TFKNESENTCQDVDECQQNPR 123 404 RLSAVNSIFLSHNNTK 124 13complement factor I gi|119392081 X X ◯ X 103 FLNNGTCTAEGK 125 177FKLSDLSINSTECLHVHCR 126 177 LSDLSINSTECLHVHCR 127 464SIPACVPWSPYLFQPNDTCIVSGWGR 128 494 LISNCSKFYGNR 129 14 cystatin Fgi|20302139 X X ◯ ◯ 84 YSVEKFNNCTNDMFLFK 130 84 FNNCTNDMFLFKESR 131 137LDDCDFQTNHTLK 132 15 desmocollin 2 gi|13435364 X X X ◯ 546SLDREAETIKNGIYNITVLASDQGGR 133 629 AINDTAAR 134 16 epithelial V-likegi|21536337 X X X ◯ 39 VLEAVNGTDAR 135 antigen 1 118 LQFDDNGTYTCQVK 13617 FAT tumor suppressor 1 gi|66346693 X X ◯ ◯ 333 AIGGIDWDSHPFGYNLTLQAK137 998 QVYNLTVR 138 1551 IVVNVSDTNDHAPWFTASSYK 139 3716QLLHKINSSVTDIEEIIGVR 140 18 fibrinogen-like 2 gi|5730075 X X ◯ ◯ 263LDGSTNFTR 141 336 LHVGNYNGTAGDALR 142 19 Fraser syndrome 1 gi|108773804X X ◯ ◯ 1107 IHTPSLHVNGSLILPIGSIKPLDFSLLNVQDQEGR 143 1503IVYNITLPLHPNQGIIEHR 144 1776 ISGSEVEELSEVSNFTMEDINNKK 145 2562 YTSYNVSEK146 2667 VIINDTEDEPTLEFDKK 147 20 growth differentiation gi|4758936 X XX ◯ 70 LRANQSWEDSNTDLVPAPAVR 148 factor 15 70 ANQSWEDSNTDLVPAPAVRILTPEVR149 21 immunoglobulin gi|148664190 (isoform 1), ◯ X X X 101 FQLLNFSSSELK150 superfamily, member 4D gi|148664211 (isoform 2) 113 VSLTNVSISDEGRisoform 1 151 22 integrin, alpha 1 gi|31657142 X X X ◯ 418 NTTFNVESTK152 883 DSCESNHNITCK 153 1113 SENASLVLSSSNQK 154 23intercellular adhesion gi|4557878 X X ◯ X 202TELDLRPQGLELFENTSAPYQLQTFVLPATPPQLVSPR 155 molecule 1 267LNPTVTYGNDSFSAK 156 24 interleukin 6 receptor gi|4504673 X X X ◯ 93SVQLHDSGNYSCYR 157 25 latent transforming growth gi|18497288 X X ◯ X 89DSCQQGSNMTLIGENGHSTDTLTGSGFR 158 factor beta binding 349RLNSTHCQDINECAMPGVCR 159 protein 3 845 DRSHCEDIDECDFPAACIGGDCINTNGSYR160 26 mucin 16 gi|83367077 X X ◯ X 12586 NTSVGLLYSGCR 161 13193KFNITESVLQGLLKPLFK 162 14363 NIEDALNQLFRNSSIK 163 14417 NGTQLQNFTLDR 16427 netrin 4 gi|93204871 X X ◯ X 56 KLWADTTCGQNATELYCFYSENTDLTCRQPK 165163 YFATNCSATFGLEDDVVKK 166 28 neuronal cell adhesion gi|81158226 X ◯ XX 223 FNHTQTIQQK 167 molecule isoform A 245, 251VISVDELNDTIAANLSDTEFYGAK 168 276 ERPPTFLTPEGNASNKEELR 169 314 EDGMLPKNR170 507 GSALHEDIYVLHENGTLEIPVAQKDSTGTYTCVAR 171 858 VNVVNSTLAEVHWDPVPLK172 29 olfactomedin related ER gi|17136143 (isoform 1), ◯ ◯ X X 85QLLEKVQNMSQSIEVLDR 173 localized protein gi|5453547 (isoform 2) 85VQNMSQSIEVLDRR 174 270 SMVDFMNTDNFTSHR 175 376 LDPVSLQTLQTWNTSYPKR 17685 QLLEKVQNMSQSIEVLDR 177 30 osteoprotegerin gi|148743793 X X ◯ ◯ 98ELQYVKQECNR 178 152 CPDGFFSNETSSKAPCR 179 178 GNATHDNICSGNSESTQK 180 289HIGHANLTFEQLR 181 31 palmitoyl-protein gi|4506031 X X X ◯ 212 GINESYKK182 thioesterase 1 (ceroid- 232 FLNDSIVDPVDSEWFGFYR 183 lipofuscinosis,neuronal 1, infantile) 32 peptidylprolyl isomerase B gi|4758950 X X X ◯148 HYGPGWVSMANAGKDTNGSQFFITTVK 184 33 plasminogen activator, gi|4505861X X ◯ ◯ 152 GTWSTAESGAECTNWNSSALAQKPYSGR 185 tissue type I 219AGKYSSEFCSTPACSEGNSDCYFGNGSAYR 186 219 YSSEFCSTPACSEGNSDCYFGNGSAYR 187483 CTSQHLLNRTVTDNMLCAGDTR 188 34 prion protein gi|122056623, X X X ◯197 QHTVTTTTKGENFTETDVK 189 gi|122056625 197 GENFTETDVK 190 gi|122056628gi|34335270, gi|4506113 35 prostaglandin H2 D- gi|32171249 X X X ◯ 51WFSAGLASNSSWLR 191 isomerase 78 SWAPATDGGLNLTSTFLR 192 36protein tyrosine gi|109633041 (isoform 1), ◯ X X X 721 KVEVEPLNSTAVHVYWK193 phosphatase, receptor gi|109633039 (isoform 2), 966DINSQQELQNITTDTRFTLTGLKPDTTYDIK 194 type, F 721 KVEVEPLNSTAVHVYWK 195957 DINSQQELQNITTDTRFTLTGLKPDTTYDIK 196 37 protein tyrosinegi|110735404 (isoform 1), X X ◯ X 410 QLTLQWEPLGYNVTR 197phosphatase, receptor gi|l10735406 (isoform 2), type, Ugi|110735402 (isoform 3) 38 seizure related 6 homologgi|6912612 (isoform 1), ◯ ◯ X X 177 LLANSSMLGEGQVLR 198 (mouse)-like 2gi|42491358 (isoform 2) 303 IVSPEPGGAVGPNLTCR 199 247 LLANSSMLGEGQVLR200 373 IVSPEPGGAVGPNLTCR 201 39 seizure related 6 homolog gi|32261332 ◯X X X 328 SVNLSDGELLSIR 202 (mouse)-like 40 seizure related 6 homologgi|148839280 (isoform 1), X ◯ X X 399, 422HLTCLNATQPFWDSKEPVCIAACGGVIRNATTGR 203 gi|148839346 (isoform 2) 436, 440IVSPGFPGNYSNNLTCHWLLEAPEGQR 204 41 serine carboxypeptidase gi|83641874,◯ ◯ X X 346 QAIHVGNQTFNDGTIVEK 205 vitellogenic-like gi|83641876  42solute carder family 39 gi|55741750 X X ◯ X 191, 198LHHHLDHNNTHHFHNDSITPSER 206 (zinc transporter), 218GEPSNEPSTETNKTQEQSDVKLPK 207 member 10 339 KDLNEDDHHHECLNVTQLLK 208 43tenascin C (hexabrachion) gi|4504549 X X ◯ X 38 QSGVNATLPEENQPVVFNHVYNIK209 327 CINGTCYCEEGFTGEDCGKPTCPHACHTQGR 210 788QTGLAPGQEYEISLHIVKNNTRGPGLK 211 1018 LNYSLPTGQWVGVQLPR 212 1034NTTSYVLRGLEPGQEYNVLLTAEK 213 1079 VKASTEQAPELENLTVTEVGWDGLR 214 1093LNWTAADQAYEHFIIQVQEAMEAAR 215 1485 LLETVEYNISGAER 216 44tissue factor pathway gi|5454114 X X ◯ X 145 YFYNNQTK 217 inhibitor 45transforming growth gi|63025222 X X ◯ ◯ 82 LRLASPPSQGEVPPGPLPEAVLALYNSTR218 factor, beta 1 46 tumor-associated calcium gi|4505059, X X ◯ X 111QCNGTSTCWCVNTAGVR 219 signal transducer 1 gi|4505057 168HRPTAGAFNHSDLDAELR 220 47 UDP-GlcNAc:betaGal gi|9845238 X X ◯ X 89LSNISHLNYCEPDLR 221 beta-1,3-N-acetyl- 173 ESWGQESNAGNQTVVR 222glucosaminyltransferase 2 48 von Willebrand factor A gi|38348304 X X ◯ ◯147 NASVPQILIIVTDGK 223 domain containing 2


2. The lung cancer differential marker glycoprotein of claim 1, whereinthe glycan is at least one glycan selected from the group consisting ofa fucosylated glycan, a high mannose-type glycan, a hybrid-type glycan,a biantennary complex-type glycan, chitin, polylactosamine, and aβ1,3-galactose epitope.
 3. The lung cancer differential markerglycoprotein of claim 2, wherein the glycoprotein is a component of adifferential diagnosis assay for small cell lung cancer or lungadenocarcinoma.
 4. The lung cancer differential marker glycoprotein ofclaim 3, wherein the glycoprotein is a component of a differentialdiagnosis assay for small cell lung cancer and is selected from thegroup consisting of neural cell adhesion molecule (NCAM1), secretograninIII, and insulin-like growth factor-binding protein-L1 (IGFBP-L1). 5.The lung cancer differential marker glycoprotein of claim 3, wherein theglycoprotein is a component of a differential diagnosis assay for lungadenocarcinoma and is fibronectin
 1. 6. A fragment of a lung cancerdifferential marker glycoprotein of claim 1 comprising at least oneasparagine residue at a glycosylation site shown in Table 1 or Table 2being glycosylated with a glycan.
 7. A method for determining lungcancercomprising detecting at least one lung cancer differential markerglycoprotein shown in Table 1 or Table 2 being glycosylated with aglycan at an asparagine residue at at least one glycosylation site shownin Table 1 or Table 2, or at least one fragment thereof, the fragmentcomprising at least one asparagine residue at a glycosylation site shownin Table 1 or Table 2 being glycosylated with a glycan, from a sampleobtained from a test subject, wherein the detection of the glycoproteinor fragment determines that the test subject suffers lung cancer.
 8. Themethod of claim 7, wherein at least one of the lung cancer differentialmarker glycoprotein or the fragment thereof are detected using at leastone glycan probe binding to the glycan.
 9. The method of claim 8,wherein the glycan probe binds to a fucosylated glycan, a highmannose-type glycan, a hybrid-type glycan, a biantennary complex-typeglycan, chitin, polylactosamine, or a β1,3-galactose epitope.
 10. Themethod of claim 8, wherein the glycan probe is a lectin, an antibody, ora phage antibody.
 11. The method of claim 10, wherein the lectin is AAL,ConA, PWM, or PNA.
 12. The method of claim 11, wherein the lung cancerdifferential marker glycoprotein is neural cell adhesion molecule(NCAM1), and the detection of the binding thereof to AAL determines thehistological type of the lung cancer as small cell cancer.
 13. Themethod of claim 11, wherein the lung cancer differential markerglycoprotein is secretogranin III, and the detection of the bindingthereof to at least one of AAL or ConA determines the histological typeof the lung cancer as small cell cancer.
 14. The method of claim 11,wherein the lung cancer differential marker glycoprotein is insulin-likegrowth factor-binding protein-L1 (IGFBP-L1), and the detection of thebinding thereof to at least one of ConA or PWM determines thehistological type of the lung cancer as small cell cancer.
 15. Themethod of claim 11, wherein the lung cancer differential markerglycoprotein is fibronectin 1, and the detection of the binding thereofto at least one of AAL or PNA determines the histological type of thelung cancer as adenocarcinoma.
 16. The method of claim 8, wherein thehistological type of the lung cancer is determined as small cell canceror adenocarcinoma on the basis of binding of the glycan probe to theglycan in at least one of the lung cancer differential markerglycoprotein or the fragment thereof, and a manner of binding at leastone of the lung cancer differential marker glycoprotein shown in Table 1or Table 2 or the fragment thereof to the glycan probe.
 17. The methodof claim 7, wherein the sample is a body fluid, a cell, or a lunglavage.
 18. The method of claim 17, wherein the body fluid is pleuraleffusion, lymph, a cell extract, sputum, or blood comprising serum,plasma and interstitial fluid.
 19. A lung cancer cell-identifyingantibody for histological staining, binding to a lung cancerdifferential marker glycoprotein listed in Table 1 or Table 2 beingglycosylated with a glycan at an asparagine residue at a glycolysationsite shown in Table 1 or Table 2, or a fragment thereof, the fragmentcomprising at least one asparagine residue at a glycosylation site shownin Table 1 or Table 2 being glycosylated with a glycan, and therebydiagnosing lung cancer.
 20. The antibody of claim 19, wherein theantibody is a determinant for a histological type of a lung cancer cell.21. The antibody of claim 20, wherein the lung cancer differentialmarker glycoprotein is neuronal pentraxin receptor, and the histologicaltype of the lung cancer cell is determined as small cell cancer.